Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.     

The elimination ofO-linked glycans from glycoproteins under non-reducing conditions

A simple procedure is described for the elimination ofO-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive -elimination ofO-glycans.     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Are pre-hospital deaths from accidental injury preventable?

OBJECTIVE--To determine what proportion of pre-hospital deaths from accidental injury--deaths at the scene of the accident and those that occur before the person has reached hospital--are preventable. DESIGN--Retrospective study of all deaths from accidental injury that occurred between 1 January 1987 and 31 December 1990 and were reported to the coroner. SETTING--North Staffordshire. MAIN OUTCOME MEASURES--Injury severity score, probability of survival (probit analysis), and airway obstruction. RESULTS--There were 152 pre-hospital deaths from accidental injury (110 males and 42 females). In the same period there were 257 deaths in hospital from accidental injury (136 males and 121 females). The average age at death was 41.9 years for those who died before reaching hospital, and their average injury severity score was 29.3. In contrast, those who died in hospital were older and equally likely to be males or females. Important neurological injury occurred in 113 pre-hospital deaths, and evidence of airway obstruction in 59. Eighty six pre-hospital deaths were due to road traffic accidents, and 37 of these were occupants in cars. On the basis of the injury severity score and age, death was found to have been inevitable or highly likely in 92 cases. In the remaining 60 cases death had not been inevitable and airway obstruction was present in up to 51 patients with injuries that they might have survived. CONCLUSION--Death was potentially preventable in at least 39% of those who died from accidental injury before they reached hospital. Training in first aid should be available more widely, and particularly to motorists as many pre-hospital deaths that could be prevented are due to road accidents.     

Restriction enzymes in cells, not eppendorfs

Restriction enzymes are essential reagents to molecular biologists, but their relevance to bacterial populations is less obvious. Most bacteria encode restriction and modification systems and these are commonly considered to be a barrier to phage infection. Current evidence also supports a more general role for them in genetic recombination.     

A PvuII polymorphism near exon 2 of the dystrophin gene

We report a PvuII polymorphism near exon 2 of the dystrophin gene with a heterozygosity frequency of 0.5.     

Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A

The human red blood cell sialoglycoprotein, glycophorin A (GpA),contains a ‘mucin-like’ extensively O-glycosylatedextracellular domain which carries the MN blood group antigens.We have revised the sites of O-glyccsylation in the extracellulardomain of GpA by automated solid-phase Edman degradation, whichallowed positive identification and quantitation of O-glycosylatedSer and Thr residues, as well as the single N-glycosylationsite. One N-linked and 16 O-linked sites were identified. Carbohydratewas absent on Ser 1, Ser14, Ser15, Ser23, Thr28 and Thr58 inGpA. We propose that the glycosyltransferases present in erythrocytesrecognize specific flanking sequences around potential O-glycosylationsites. All 16 O-glycosylation sites are explained on the basisof four motifs. Three motifs are associated with Thr-glycosylation:Xaa—Pro—Xaa—Xaa where at least one Xaa = Thr;Thr—Xaa—Xaa—Xaa where at least one Xaa = Thr;Xaa—Xaa—Thr—Xaa where at least one X = Argor Lys. The fourth motif is associated with Ser-glycosylation:Ser—Xaa—Xaa—Xaa where at least one Xaa = Ser.These simple rules explain the glycosylation (or lack of it)on 21 of 22 Ser/Thr in the extracellular domain of GpA. glycophorin A O-glycosylation motif solid-phase Edman degradation