Study of human chromosome V

Summary The induction of fragile sites on human chromosomes has been demonstrated under various conditions that cause thymidylate stress, including exposure to uridine. In this study, we examined common fragile site expression by initially exposing peripheral lymphocytes to uridine, followed by repair of the fragile sites with media containing various concentrations of thymidine. Lymphocytes were cultured in medium 199 with 2 mM uridine. At 0.5, 1, 2, 3, 8, 10, 12, and 18 h before harvest, the uridine medium was removed and replaced by medium containing thymidine at various concentrations. Our results demonstrate that the effect of uridine on chromosome fragility can be reversed by low concentrations of thymidine (2 M up to 200 M) and the rescuing effect of thymidine can be achieved if the cells were treated prior to 2–3 h before harvest. No repair was found if thymidine was added to culture within 2 h prior to harvesting, suggesting that packing of chromosomes is also an important factor in the expression and repair of fragile sites.     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Intercellular structure in a many-celled magnetotactic prokaryote

A many-called magnetotactic prokaryote obtained from brackish water was observed to possess intercellular connections at points of contact between the outer membranes of constituent cells. Each aggregate organism consisted of 10 to 30 individual Gram-negative cells containing material with the appearance of poly--hydroxybutyrate and magnetosomes of unusual arrangement, structure and composition. The aggregate, which possessed prokaryotic-type flagella arranged at the outwards surfaces of each cell, showed motility indicative of co-ordination between individual component cells. These results suggest that this organism could be a multicellular prokaryote.     

Nerve growth factor: Cellular localization and regulation of synthesis

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.     

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP₁, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and -mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP₁ is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.Abbreviations PMSF phenylmethyl sulfonylfluoride - TCA trichloroacetic acid - DTT dithiotreitol     

Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: Characterization of permissive and inducible C2 myoblasts

Using subcloning and manipulations of culture conditions we have isolated from the mouse myogenic cell line C2 a variant cell line that we named inducible. Unlike the progenitor cells that are referred to as permissive, inducible myoblasts differentiate poorly in Dulbecco modified Eagle medium plus fetal calf serum (FCS) and require the presence of insulin at a high concentration (1.6 10(-6) M) or insulin-like growth factor I (IGFI) at a lower concentration (2.5 10(-8) M) to differentiate. Permissive and inducible myoblasts fail to differentiate when grown in MCDB202 medium plus 20% FCS, even after a prolonged arrest in G1 phase. This shows that an arrest in G1 is in itself insufficient to trigger terminal differentiation. Both cell types also exhibit distinct patterns of accumulation of muscle mRNAs corresponding to sarcomeric actins and myosin light chain MLC1A. The possibility that these two cell lines might represent two different stages of the progression of myoblasts toward terminal differentiation is discussed.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.