Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.     

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.     

A second gene (qutH) within the Aspergillus nidulans-quinic-acid utilisation gene cluster encodes a protein with a putative zinc-cluster motif.

A sequence of 3299 nt, contiguous with the previously sequenced quinate permease-encoding (qutD) gene and encompassing the dehydroshikimate dehydratase-encoding (qutC) gene, has been determined. Northern-blot analysis detected (i) a quinate-inducible mRNA of the expected size for the qutC gene, and (ii) a quinate-inducible mRNA of 1.45 kb divergently transcribed away from qutC towards qutD. Computer-aided sequence analysis identified an ORF of 1047 nt corresponding to the qutC gene encoding dehydroshikimate dehydratase. In addition, a genetically uncharacterized 1188-nt gene, designated qutH and containing a putative intron of 61 nt, was identified between qutC and qutD. The inferred protein sequence encoded by qutH contains a putative 'zinc cluster' motif and has a low (16%) but significant similarity with the DNA-directed DNA polymerase of hepatitis B virus. The results are interpreted as being consistent with the view that the qutH gene encodes a DNA-binding protein, possibly involved in the regulation of genes essential for the utilisation of protocatechuic acid.     

The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks.

The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family.

A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S. cerevisiae. Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein. Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation. One was CDC2-HS, and the second was the human homolog of Xenopus CDK2. Cyclin E produced in E. coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells. The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.     

Quantity and quality of embryos collected from mice passively immunized against estradiol

The objective of this study was to determine the effects of passive immunization against estradiol on the occurrence and timing of estrus, ovulation and fertilization rates and on early embryonic development in mice. Swiss Webster fermale mice were randomly assigned to one of the three treatment groups to be injected with 0.1 ml saline (control; n=15), 0.1 ml anti-estradiol antisera (high dose: n=17) or 0.1 ml antiestradiol antisera diluted 1:10 with saline (low dose; n=17) at seven weeks of age. Immediately after injection mice were placed with males and observed daily for the presence of vaginal plugs for 10 d. Three days after vaginal plugs were observed, mice were terminated and the uteri were removed and flushed to determine the number and quality of unfertilized ova and embryos. No differences were observed in the timing of vaginal plug formation, the proportion of mice with vaginal plugs, or the mean number of unfertilized ova or embryos collected from each treatment group. However, the proportion of excellent or good quality embryos was reduced in the high dosage treatment. It was concluded from this study that passive immunization of mice against estradiol did not increase the number of embryos obtained from mice and that a high dosage of antisera against estradiol reduced embryo quality. These results provide evidence that alterations in levels of estradiol may adversely affect embryonic development.     

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.     

A protoplast to plant system in roses

High yields of protoplasts were isolated from embryogenic suspension cultures of Rosa persica x xanthina and Rosa wichuraiana using an enzyme mixture comprising 20 g l^-1 cellulase Onozuka R10, 1 g l^-1 Pectolyase Y-23 and 10 g l^-1 hemicellulase. Agarose-immobilized protoplasts gave the most consistent growth at a plating density of 5×10⁴ protoplasts ml^-1 on the basic medium of Kao & Michayluk (KM8p) containing 2 mg l^-1 naphthaleneacetic acid and 1 mg l^-1 benzylaminopurine. At 25°C in the dark, 0.004% of R. persica x xanthina protoplasts developed into colonies. Using similar culture conditions, but with a plating density of 9×10⁴ protoplasts ml^-1, 0.017% of R. wichuraiana protoplasts developed into colonies. On transfer of R. persica x xanthina colonies to Schenk & Hildebrandt's medium containing 3 mg l^-1 2,4-dichlorophenoxyacetic acid, globular and later stage embryos were formed. Approximately 30% of these embryos developed into plantlets on transfer to basal Schenk & Hildebrandt's medium. Further development of the plantlets took place on cellulose plugs (Sorbarods) soaked in Murashige & Skoog's medium containing 0.05 mg l^-1 naphthaleneacetic acid, 0.05 mg l^-1 indole-3-butyric acid and 0.1 mg l^-1 benzylaminopurine. Rose breeding is now open to the full range of in vitro genetic manipulation techniques involving protoplast technology.