Embryogenesis of the free-living soil nematodeCaenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock-i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. The cell lineages reported here were analyzed from tapes of two eggs recording the complete development until the animals started moving inside the egg shell. In addition, embryogenesis was recorded and analyzed in 18 other individuals from fertilization to the 30-(10 cases), the 54-, 60-, 75-, and 87-(3 cases), and the 100-(2 cases) cell stages, respectively. We also analyzed the E-cell lineage in six more individuals to make certain that all 8 E-cells divide in going to 16. After recording was terminated, it was ascertained that, under the cover glass, all 26 animals hatched and moved normally.

In addition to their well defined role in presentation of processed antigen on the cell surface, class II molecules are able to transduce signals into the cell after binding of ligands. The cytoplasmic regions of class II molecules might function as docking sites for as yet unidentified proteins that are components of this signalling pathway. Here we report on two putative HLA class II associated proteins (PHAPI and PHAPII) which have been purified from the cytosolic fraction of the human lymphoblastoid B-cell line H2LCL using an affinity matrix composed of the synthetic biotinylated cytoplasmic region of the DR2 alpha chain immobilized on avidin agarose. The sequence obtained for PHAPI revealed a novel primary structure with a leucine/isoleucine rich N-terminal region. Protein data and the cDNA sequence obtained for PHAPII agree with the cDNA sequence of SET that has been described recently. Both PHAPI and PHAPII have an extended highly acidic C-terminal region. Based on their primary structure we speculate that PHAPI and PHAPII are involved in the generation of intracellular signalling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules.

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (−)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3′,5′- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-α, -βII, -δ, -ε, and -ζ were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-ε for 48 h prevented stimulation of CFTR with (−)-epinephrine, reduced PKC-ε activity in unstimulated cells by 52.1%, and decreased PKC-ε mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-ε and antisense oligonucleotide to PKC-δ and -ζ did not alter (−)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-ε.

Thymidine kinase positive (TK+) N type cell lines that had been transformed by spleen focus-forming virus were established by transformation with NB tropic Friend virus complex. Thymidine kinase deficient (TK-) cell clones were isolated. Some of these cell clones release 1000-to 100,000-fold reduced amounts of Friend virus complex as compared to the TK+ parental cell clone. TK-clones were grown in medium without BrdUrd. Some of these TK-clones can be induced to release endogenous helper virus and transforming spleen focus-forming virus on reexposure to 10-10-4 M BrdUrd. BrdUrd inhibits the replication of C-type tumor viruses (1), whereas in some instances BrdUrd or IdUrd can induce endogenous C-type virus (2-4). In order to study the action of BrdUrd on virus replication and its role in the induction of endogenous virus we have used Friend virus (FV) infected and transformed DBA-2 mouse spleen cells in culture. The FV complex consists of two components, the lymphatic leukemia helper virus (LLV-F) and the replication-defective erythroid cell-transforming spleen focus-forming virus (SFFV-F) (5, 6). The RNA of the two virus types is presumably different in size and structure (7). We have established various cell lines transformed by the SFFV-F (8, 9) which, after another thymidine analogue, can be phosphorylated to azidothymidine triphosphate. However, the presence of an No group at the 3' terminal of the deoxysugar interferes with esterification, so presumably azidothymidine can only be added terminally to the growing DNA strand. Both azidothymidine as well as BrdUrd can be used to decrease the virus titer of BrdUrd-sensitive erythroleukemic cells. We have isolated several BrdUrd-resistant clones by treatment of sensitive cells with high doses of BrdUrd (11) and have shown that some of these clones have either decreased or barely detectable thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.75) activity (11). Furthermore, they have a 108-to 104-fold decreased virus titer. The decrease in C-type virus release can also be shown by electron microscopy.A temporary thymidine kinase activation is observed if BrdUrd is added to some virus negative lines. The same cells release large numbers of endogenous C-type and spleen focusforming virus on exposure to BrdUrd but not to azidothymidine. The RNA tumor virus which is induced by BrdUrd has mainly N-type host range properties unlike the original NB type FV complex which has been used for the transformation of our erythroid cell lines. Thymidine kinase induction and virus induction are always correlated. We have independent evidence (Ostertag, Crozier, and Swetly, unpublished)

Hyperosmotic stress activates Na+-K+-2Cl- cotransport (NKCC1) in secretory epithelia of the airways. NKCC1 activation was studied as uptake of 36Cl or 86Rb in human tracheal epithelial cells (HTEC). Application of hypertonic sucrose or NaCl increased bumetanide-sensitive ion uptake but did not affect Na+/H+ and Cl-/OH-(HCO3-) exchange carriers. Hyperosmolarity decreased intracellular volume (Vi) after 10 min from 7.8 to 5.4 microl/mg protein and increased intracellular Cl- (Cl-i) from 353 to 532 nmol/mg protein. Treatment with an alpha-adrenergic agent rapidly increased Cl-i and Vi in a bumetanide-sensitive manner, indicating uptake of ions by NKCC1 followed by osmotically obligated water. These results indicate that HTEC act as osmometers but lose intracellular water slowly. Hyperosmotic stress also increased the activity of PKC-delta and of the extracellular signal-regulated kinase ERK subgroup of the MAPK family. Activity of stress-activated protein kinase JNK was not affected by hyperosmolarity. PD-98059, an inhibitor of the ERK cascade, reduced ERK activity and bumetanide-sensitive 36Cl uptake. PKC inhibitors blocked activation of ERK indicating that PKC may be a downstream activator of ERK. The results indicate that hyperosmotic stress activates NKCC1 and this activation is regulated by PKC-delta and ERK.