Temporary complementation of temperature-sensitive mutants of the cell cycle by transfection with a wild-type or a mutant cDNA of ADP/ATP translocase

A number of cell-cycle-specific temperature-sensitive (ts) mutants have been isolated from animal cells, especially Syrian hamster cells. These ts mutants, like cell cycle ts mutants of yeast, can be complemented by specific genes, some of which have been molecularly cloned. We have isolated a cDNA clone that complements TK-ts13 cells, but only temporarily. This clone, called B1, differs from a previously isolated clone (Sekiguchi et al.: EMBO Journal 7:1683-1687, 1988) that specifically complements ts13 cells. In addition, B1 also complemented temporarily three other ts mutants of the cell cycle, tsAF8, ts694, and ts550C cells. These mutants have different mutations since, in cell fusion experiments, they complement each other. Sequencing of the B1 cDNA clone revealed that it was a mutant of human ADP/ATP translocase in which some human sequences at the 5' end have been replaced by SV40 sequences. The wild-type translocase was less effective but could still increase the survival time of cell cycle ts mutants at the restrictive temperature. Using the polymerase chain reaction, it was possible to demonstrate that the B1 plasmid is expressed in TK-ts13 cells undergoing temporary complementation.     

High apoplastic solute concentrations in leaves alter water relations of the halophytic shrub, Sarcobatus vermiculatus

Predawn plant water potential (Psi(w)) is used to estimate soil moisture available to plants because plants are expected to equilibrate with the root-zone Psi(w). Although this equilibrium assumption provides the basis for interpreting many physiological and ecological parameters, much work suggests predawn plant Psi(w) is often more negative than root-zone soil Psi(w). For many halophytes even when soils are well-watered and night-time shoot and root water loss eliminated, predawn disequilibrium (PDD) between leaf and soil Psi(w) can exceed 0.5 MPa. A model halophyte, Sarcobatus vermiculatus, was used to test the predictions that low predawn solute potential (Psi(s)) in the leaf apoplast is a major mechanism driving PDD and that low Psi(s) is due to high Na+ and K+ concentrations in the leaf apoplast. Measurements of leaf cell turgor (Psi(p)) and solute potential (Psi(s)) of plants grown under a range of soil salinities demonstrated that predawn symplast Psi(w) was 1.7 to 2.1 MPa more negative than predawn xylem Psi(w), indicating a significant negative apoplastic Psi(s). Measurements on isolated apoplastic fluid indicated that Na+ concentrations in the leaf apoplast ranged from 80 to 230 mM, depending on salinity, while apoplastic K+ remained around 50 mM. The water relations measurements suggest that without a low apoplastic Psi(s), predawn Psi(p) may reach pressures that could cause cell damage. It is proposed that low predawn apoplastic Psi(s) may be an efficient way to regulate Psi(p) in plants that accumulate high concentrations of osmotica or when plants are subject to fluctuating patterns of soil water availability.     

Abundance and biogeography of tintinnids (Ciliophora) and associated microzooplankton in the Southwestern Atlantic Ocean

Absolute abundances of foraminifers, polycystine and phaeodarian radiolarians, tintinnids, pteropods and early crustacean larvae and moults were assessed in a collection of 57 vertically stratified (0-100 m) net microplankton samples from 22 stations located between 34 and 58S (along 51-56°W), covered on 8-16 November 1994. Tintinnids were identified to species and measured in order to estimate their biomass from biovolume to carbon conversions. The distribution of the microzooplanktonic groups assessed was irregular and patchy, both geographically and vertically, and their abundances were characteristic of oceanic low to medium productivity environments. Tintinnid biomass was also generally low (0.05-0.40 g Cl^-1). With the exception of the tintinnids, associations between microzooplanktonic numbers and chlorophyll a were generally loose. Eighty-eight tintinnid taxa were recorded, yet only five accounted for 53% of the specimens identified. Multivariate (cluster) analysis of tintinnid specific distribution patterns clearly showed several distinct zones. From north to south, these are: Transition Zone (TZ), with three subzones, TZ north (34°S-38°S), TZ central (39°44S-44°S) and TZ south (46°S); Subantarctic Zone (SZ; 48-55°S); Polar Front Zone (55°30S); Antarctic Zone (AZ; 58-59°36S). Each of these was characterized by distinct tintinnid assemblages, abundance and biomass. With few exceptions, tintinnid cells were fairly evenly distributed throughout the upper 50 m. Taxonomic composition usually changed little with depth. Mean population depths were calculated for a subset of 35 tintinnids; 29 of these dwell preferably above 40 m. The spatial distribution of tintinnid species richness showed a more or less gradual decrease from north to south. Specific diversity and equitability generally increased with depth, and were higher in antarctic waters than the southern transitional and subantarctic ones; this trend is tentatively attributed to higher water column vertical stability south of the Polar Front.      

Transdifferentiation and regeneration in vitro

Striated muscle tissue and endoderm can be isolated from the anthomedusa Podocoryne carnea. The isolates are uncontaminated by other cell types and can be cultivated in artificial seawater for months without undergoing autonomous regeneration. However, if the endoderm is combined with collagenase-treated striated muscle, a regeneration process is initiated which leads to the formation of the sexual and feeding organ (manubrium) of the medusa. The original endoderm and striated muscle are replaced in the regenerate by at least seven new cell types, including gametes. Labeling experiments with [³H]thymidine and experiments in which mitosis is inhibited in either the striated muscle or the endoderm with mitomycin C demonstrate that the striated muscle is able to transdifferentiate into all the cell types found in the regenerate. With the possible exception of ectodermal smooth muscle this statement is also valid for the endoderm.     

A kinetic and clonal analysis of heterogeneity in K562 cells

Expression of markers of differentiation was measured in a clone of the continuous cell line K562, derived originally from the cells of a patient with leukemia. Three of the markers were lineage specific, R18 for erythropoiesis and 80H.5 and My-1 for granulopoiesis. The fourth marker was the self-renewal capacity of clonogenic cells. The markers were measured as a function of time in pooled colonies from day 2 to day 12, and at a point of time in individual colonies. Evidence of an orderly pattern of marker appearance and disappearance was not seen. Rather, their expression appeared to occur at random during growth.     

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.