Sweat Chlorides in Salt-Deprived Cystic Fibrosis Heterozygotes

Sweat chlorides of 10 sets of parents of children with cystic fibrosis and 11 controls were studied in an attempt to develop a test for the diagnosis of cystic fibrosis heterozygotes by subjecting both the parents and controls to a low sodium diet and comparing sweat chloride values as the diet progressed. It was hoped that the sweat chloride levels of the parents, the heterozygotes, would remain stationary throughout the diet, since their children, the homozygotes, reveal this finding under similar conditions of salt deprivation. The sweat chloride levels of the controls, because of effects of aldosterone, were expected to decrease steadily from the commencement of the diet to its termination.A decrease in sweat chloride values of similar magnitude was found in both parents and controls as the diet continued. It is concluded that the study of sweat electrolyte levels in salt-deprived subjects is of no value in the diagnosis of cystic fibrosis heterozygotes.     

Paraspinibarbus,a new genus of cyprinid fishes from the Red River basin

Paraspinibarbus, a new and monotypic genus, is erected forSpinibarbus macracanthus Pellegrin et Chevey, 1936, a cyprinid from the Red River basin. It is characterized by a procumbent predorsal spine and a very thick lower lip with a continuous postlabial groove.Balantiocheilus hekouensis Wu, 1977 is a junior subjective synonym ofParaspinibarbus macracanthus. A lectotype is designated forS. macracanthus.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Bradykinin-induced changes in phosphoinositides, inositol phosphate production and intracellular free calcium in cultured bovine aortic endothelial cells

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10(-7)M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action. Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic acid (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate. The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 +/- 21 nM to 462 +/- 27 nM. The Ca2+ peak was at 7.0 +/- 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+. These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plant regeneration of wildGlycine species from suspension culture-derived protoplasts

Protoplasts isolated from four-week old cell suspension cultures ofGlycine canescens F. J. Herm andG. clandestina Wendl. were cultured in 8P or modified 8P to a multicellular stage. Colonies of 0.5 to 1.0 mm diameter were transferred to solid media for callus growth and regeneration. Callus consisted of friable masses with compact green nodular areas. Organogenesis of both species occurred primarily from the green nodular areas. Shoot buds ofG. clandestina did not mature, but shoots ofG. canescens proliferated on MS medium, with B5 vitamins, 0.33 mgL^–1 each BA, KN, ZN, and 0.15 mgL^–1 NAA. Shoots failed to root after multiple subcultures on four different rooting media.In vitro grafting ofG. canescens scions ontoG. max root stocks allowed plants to be transferred to soil. An overall protoplast division efficiency of 48% was achieved with moderately efficient shoot regeneration inG. canescens. Division efficiencies forG. clandestina were lower (11%). Refinements of this protocol should result in high efficiencies of regeneration which would allowin vitro manipulations of these wild soybean relatives at the single cell level and would make the derivation of somatic hybrid plants possible within the genusGlycine.Abbreviations BA 6-Benzyladenine - KN kinetin - ZN Zeatin - NAA Napthaleneacetic acid - 2,4-D 2–4-dichlorophenoxyacetic acid - PIC Picloram - CH casein hydrolysate - Gln glutamine - Met methionine - MES 2[N-morpholine] ethanesulfonic acid     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

The effect of diabetes mellitus on aortic prostanoid synthesis and serum cholesterol levels in the rat fed a high cholesterol diet

The effect of diabetes mellitus on serum cholesterol and aortic microsomal prostanoid synthesis was studied in cholesterol fed male Lewis rats. Normal, diabetic and diabetic rats treated with pancreatic islets were divided into three diet subgroups, control diet, control +2% cholesterol for 8 weeks and control +2% cholesterol diet for 16 weeks. Serum glucose levels were elevated three-fold in the diabetic group compared to normal. Treatment with islets restored serum glucose to normal levels in diabetic rats. The 2% cholesterol diet did not significantly alter serum glucose levels in any of the groups. Body weights in the diabetic group were significantly lower than normal or diabetic rats treated with islets. Feeding 2% cholesterol for 16 weeks significantly increased weight in normal and islet treated diabetic rats but not in the diabetic group. Aortic microsomal prostanoid synthesis was similar in all experimental groups with 6-keto-PGF1 alpha (PGI2 metabolite) being the major product synthesized in all groups. Aortic microsomal prostanoid levels were not altered by the 2% cholesterol diet. Serum cholesterol levels increased 14-fold in the diabetic group which returned to the normal level in the diabetic animals treated with islets. These data show that diabetes does not alter aortic microsomal prostanoid levels in the rat. However, diabetes significantly increased serum cholesterol levels which were reversed by islet transplantation.