Reversal of cellular polarity and early cell-cell interaction in the embryos of Caenorhabditis elegans

During early embryogenesis of Caenorhabditis elegans the serial stem cell-like cleavages of the germ line cells P0-P3 generate a number of somatic founder cells with different developmental potentials. Observations on partial embryos show that in the first two of these unequal divisions in the germ line the somatic daughter cell comes to lie anterior to the new germ line cell. In the following two, however, the somatic daughter cell comes to lie posterior to the new germ line cell, suggesting a reversal of polarity in the germ line. By the use of a laser microbeam, egg fragments can be extruded from young embryos; the fragments often cleave like partial twins. Depending on whether the fragment is derived from the posterior region of the uncleaved zygote P0 or its daughter P1, the mirror image duplications that are generated are joined at their larger soma-like cells or at their smaller germ line-like cells, respectively. This result is best explained as a reversal of polarity taking place in the germ line cell P2. This notion is strengthened by the finding that partial embryos derived from the posterior region of the P2 cell in late interphase do not undergo stem cell-like (i.e., unequal) cleavages in contrast to those derived from P0 or P1. Finally, an apparent early cell-cell interaction is described which is inconsistent with the classical notion of "mosaic" nematode development: removal of the germline cell P2 results in an altered developmental pattern of its somatic sister cell EMS. A working model is presented linking reversal of polarity and cell-cell interaction and offers an explanation for the unique behavior of the EMS cell in normal development.     

Molecular basis of an isogeneic anti-idiotypic response.

The nucleotide sequences of the variable region genes expressed in the heavy and light chains of six isogeneic anti-idiotope antibodies recognizing idiotopes on two closely related antibodies with specificity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were determined. In two independently derived anti-idiotope cell lines the same or strongly homologous V kappa, VH and D region genes had originally been rearranged. The two lines express long and partly homologous N sequences (presumed to be not of germ line origin) at the border of D, resulting in CDR3s of unusual length. An unusually long CDR3, partly encoded by N sequences, is also present in the heavy chain of a third anti-idiotope antibody. The VH regions of the three remaining anti-idiotope antibodies originate from a single VH gene which belongs to the same VH group as the VH genes expressed in the other anti-idiotopes. Two of these antibodies, expressing similar V, D and J elements, had been isolated from the same mouse and appear to have diverged from the same B cell precursor by at least two rounds of somatic mutation. Somatic point mutations have occurred in most, if not all anti-idiotope V region sequences. In two instances somatic mutations in J increase the structural homology between anti-idiotopes. The anti-idiotypic response in this system is thus genetically restricted and may depend upon the selection of non-germ line sequences, suggesting an explanation for the low frequency at which anti-idiotope antibodies are expressed in this system.     

Biochemically labelled human lymphoblastoid cell line. I. Its karyotypic, growth and physiological characteristics

A study was made of the properties of human lymphoid cell line RPMI-6410 derived from peripheral blood of a patient with acute myeloblastic leukaemia. The lymphoblastoid cell line was found to be resistant to 5-brom-deoxyuridine and to have a low thymidine kinase activity. The modal chromosome number for RPMI-6410 is 46-47 XY. The karyotype includes marker chromosomes: two large submetacentrics --M1 and M2, and two small acrocentrics--M3 and M4. Ways of marker chromosome formation are discussed. The properties of RPMI-6410 line make it possible to use it for somatic cell hybridization, in particular, for obtaining hybridoma synthesizing human monoclonal antibodies.     

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.     

Probing spermatogenesis in Drosophila with P-element enhancer detectors.

Formation of motile sperm in Drosophila melanogaster requires the coordination of processes such as stem cell division, mitotic and meiotic control and structural reorganization of a cell. Proper execution of spermatogenesis entails the differentiation of cells derived from two distinct embryonic lineages, the germ line and the somatic mesoderm. Through an analysis of homozygous viable and fertile enhancer detector lines, we have identified molecular markers for the different cell types present in testes. Some lines label germ cells or somatic cyst cells in a stage-specific manner during their differentiation program. These expression patterns reveal transient identities for the cyst cells that had not been previously recognized by morphological criteria. A marker line labels early stages of male but not female germ cell differentiation and proves useful in the analysis of germ line sex-determination. Other lines label the hub of somatic cells around which germ line stem cells are anchored. By analyzing the fate of the somatic hub in an agametic background, we show that the germ line plays some role in directing its size and its position in the testis. We also describe how marker lines enable us to identify presumptive cells in the embryonic gonadal mesoderm before they give rise to morphologically distinct cell types. Finally, this collection of marker lines will allow the characterization of genes expressed either in the germ line or in the soma during spermatogenesis.     

Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell nuclear transfer

Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

HLA antigen expression at the single cell level on a K562 X B cell hybrid: an analysis with monoclonal antibodies using bacterial binding assays

The influence of different genetic environments on the expression of HLA complex-controlled antigens has been investigated using cell lines with various defects in the synthesis of these molecules and a somatic cell hybrid derived from them. A very sensitive bacterial binding assay allowing simultaneous evaluation of the morphology of a given cell and the quantity of a surface molecule has been developed for these studies. The fetal erythroid cell line K562, the Burkitt's lymphoma-derived cell line DAUDI, and their hybrid DUTKO1 have been employed. K562 and the hybrid, but not DAUDI, expressed HLA-A,B,C heavy chains as detected by the monoclonal antibody W6/32.HL, while two monoclonal antibodies (TU48 and 2BC4) against the supertypic specificities HLA-Bw4 and Bw6 showed no reactivity. The presence of human Ia-like antigens on the cell surfaces was investigated with a panel of eight monoclonal antibodies. K562 cells were completely unreactive, and DAUDI cells gave the expected positive reaction, but about 1% or less of the cells in the DUTKO1 population appeared to express these antigens as well. We discuss possible reasons for the failure to detect HLA-B antigens with monoclonal antibodies and the lack of complete "dominance" of the K562 genome in the hybrid cell line.     

Localization of the t(2;13) breakpoint of alveolar rhabdomyosarcoma on a physical map of chromosome 2.

A characteristic translocation t(2;13)(q35;q14) has been previously identified in the pediatric soft tissue tumor alveolar rhabdomyosarcoma. We have assembled a panel of lymphoblast, fibroblast, and somatic cell hybrid cell lines with deletions and unbalanced translocations involving chromosome 2 to develop a physical map of the distal 2q region. Twenty-two probes were localized on this physical map by Southern blot analysis of the mapping panel. The position of these probes with respect to the t(2;13) rhabdomyosarcoma breakpoint was then determined by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2 and by analysis of somatic cell hybrid clones derived from an alveolar rhabdomyosarcoma cell line. We demonstrate that the t(2;13) breakpoint is situated within a map interval delimited by the distal deletion breakpoint in fibroblast line GM09892 and the t(X;2) breakpoint in somatic cell hybrid GM11022. Furthermore, from a comparison of our data with the linkage map of the syntenic region on mouse chromosome 1, we conclude that the t(2;13) breakpoint is most closely flanked by loci INHA and ALPI within this map interval.     

Evidence of selective processing of immunodominant epitopes in virally infected cells

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies

Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce Ig M-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase(AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line(DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus.The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.     

Direct involvement of surface IA/E molecules during B cell maturation using an antigen-specific B cell clone

TP67.14 is a subclone of a resulting B cell hybridoma established by somatic hybridization between splenic B cells of A/J mice immunized with TNP-LPS and 2.52 M, a HAT medium-sensitive mutant of a B cell line; it expresses IgM, B220, IAk, and IEk on the cell membrane and also possesses a receptor molecule for TNP on its surface derived from TNP-reactive B cells of A/J mice used for cell fusion. As shown previously, TP67.14 could be induced to generate a significant amount of anti-TNP antibodies when treated with TNP-conjugated protein such as TNP-BSA and TNP-keyhole limpet hemocyanin without T cell help as well as LPS. Our study was undertaken to investigate direct involvement of surface MHC class II molecules on B cells during B cell maturation by analysis with this Ag-specific B cell clone. The data demonstrate that mAb against IAk and IEk molecules, but not IAd and H-2k, markedly inhibited the differentiative effects of LPS on TP67.14. In contrast, both antibodies specifically augmented the secretion of anti-TNP antibodies by TP67.14 treated with TNP-BSA, although these antibodies alone failed to induce the generation of anti-TNP antibodies. Interestingly, TP67.14 significantly differentiated into anti-TNP antibody secreting cells when incubated with TNP-conjugated monoclonal anti-IAk or anti-IEk antibodies alone; this differentiative effect was much greater than that of TNP-conjugated anti-IAd mAb or purified mouse IgG under the same conditions. Our result suggests that surface IA/E molecules on B cells may be directly involved in a transductional signal for B cell maturation mediated by the cross-linkage of receptor molecules on B cells with Ag.     

Analysis of somatic cell H-2 variants to define the structural requirements for class I antigen expression

We have taken the approach of producing somatic cell variants with altered H-2 products to study the structural requirements for cell surface expression of class I histocompatibility molecules. H-2 antigen variants generated by chemical mutagenesis of a cell line expressing the H-2b haplotype were first selected with alloantisera for their loss of H-2Kb expression, and then were analyzed by radioimmunoassay for the appearance of intracellular Kb antigen. For one such variant (69.9.15), whereas the H-2Kb antigen was absent from the cell surface as assayed by antibody-mediated complement-dependent cytotoxicity, an H-2Kb molecule was detected within the cell lysate as confirmed by direct immune precipitation with Kb-specific monoclonal antibodies. The product had an altered antigenic phenotype, since it reacted with only two anti-Kb monoclonal antibodies (Y-3 and EH-144) and not with a third (5F1.2). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the beta 2 microglobulin-associated, intracellular H-2Kb heavy chain to be slightly smaller in Mr than the H-2Kb of the parental cell line. Hybridization analysis revealed the Kb gene from the variant to be without gross alterations, and furthermore, identified a Kb mRNA species that was identical in size to wild-type Kb mRNA. Because complementation was not observed after somatic cell fusion of variant cells with BALB/c splenocytes, it appeared that the alteration in Kb expression was due to a cis-acting defect. In addition, DNA-mediated gene transfer of the wild-type Kb gene into the variant cell line resulted in expression of the Kb antigen on the cell surface, thus confirming that the defect in expression of the mutant Kb product was not due to other factors in the 69.9.15 cell line. Such findings are consistent with the conclusion that stable H-2Kb surface-negative somatic variants can arise due to limited alterations in the Kb gene, resulting in the synthesis of a class I molecule that is expressed only as an intracellular product.     

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.     

Germ-line affinity and germ-line variable-region genes in the B cell response

The predominance of germ-line genes in IgM expression was evaluated from the nucleotide sequences of mRNA, derived from 10 hybridoma cell lines, coding for the VH and VL regions of anti-5-dimethylaminonaphthalene-1-sulfonyl (anti-Dns) IgM antibody. At least six germ-line VH gene segments distributed among four families are used in this response. Seven of the 10 independently rear-ranged VH genes were identified as germ line, with the other three possibly germ line. In all of them the D and JH portions retained the germ-line sequences of the D and JH segments from which they were derived. Maximum diversity was found in the D segments and the use of noncoded nucleotides at the VH-D and D-JH junctions. Of the eight cell lines expressing the lambda light chains, all were germ line and involved the three subtypes. Maximum affinity for the homologous ligand was found among the seven cell lines identified as expressing germ-line gene segments. Thus any somatic mutation among the remaining 3 cell lines did not provide enhanced affinity and the observed affinity of each cell line can be described as germ-line affinity. It is further suggested that the anti-Dns selectivity of the IgM antibodies is associated primarily with the CDR3 regions.     

Production and characteristics of a Djzungarian hamster cell line (DX-TK-) resistant to 5-bromodeoxyuridine]

New biochemically marked Djungarian hamster cell line (DX-TK-) was established. These cells are resistant to 5-bromodeoxyuridine (25 mkg/ml) and deficient in thymidine kinase activity (TK-). Due to this biochemical defect they have lost the ability to grow in HAT medium. DX-TK- cells are malignant. They grow as tumours after the inoculation to newborn Djungarian hamsters. Tumorigenecity of DX-TK- cells was decreased as compared with the parent TK+ cell line. DX-TK- cell line is a hypodiploid cell culture (26 chromosomes) with 7 chromosome markers easily identified by means of G-band staining. This line is a new model for somatic cell genetic experiments, particularly for somatic cell hybridization.     

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.     

A new murine IgG1 anti-Tn monoclonal antibody with in vivo anti-tumor activity

The Tn antigen (GalNAc α-O-Ser/Thr) is heterogeneously synthesized by a variety of tumors and contains an epitope defined by lectins and antibodies as a cluster of αGalNAc carbohydrates synthesized within a peptide sequence, which is rich in serine and/or threonine. The Tn antigen has been utilized as a target in vaccine experiments and also used as a biomarker for prognosis of different cancer forms. In this paper, we present a new monoclonal antibody, GOD3-2C4, with the clear hallmarks of an anti-Tn antibody. It was generated through somatic cell hybridization after immunization of a mouse with a tumor cell line and a Tn carrying mucin. The antibody recognizes synthetic Tn antigen and binds breast, colon, lung, ovarian and pancreas cancer. The GOD3-2C4 antibody has antibody-dependent cellular cytotoxicity activity against Jurkat cells in vitro, and for the first time, it can be shown that an anti-Tn antibody has a significant in vivo effect on a human cancer cell line grown as a xenograft in severe combined immunodeficiency mice.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.