The constitution and properties of phosphorylated and unphosphorylated C-terminal fragments of progastrin from dog and ferret antrum

Antibodies to the extreme C-terminal tryptic (nona-) peptide fragment of porcine progastrin have been used in radioimmunoassay to identify progastrin fragments in dog, ferret and pig antral mucosa extracts and to monitor their purification. In addition to previously characterised phosphorylated and unphosphorylated C-terminal tryptic peptides of porcine progastrin a minor form corresponding to the C-terminal octapeptide (i.e. des-Ser C-terminal nonapeptide) was isolated and characterised. The latter form together with phosphorylated and unphosphorylated forms of the nonapeptides were also isolated and chemically characterised from dog antrum, and the unphosphorylated nonapeptide was characterised from ferret antrum. The primary amino acid sequences of the dog, ferret and pig nonapeptides were identical. In ferret the unphosphorylated nonapeptide predominated, and in dog the phosphorylated form predominated; in pig both forms of the nonapeptide were well represented. Intact progastrin was identified in gel filtration eluates of extracts of all 3 species, but occurred only in relatively low concentrations. The nonapeptides did not stimulate acid secretion in the conscious gastric fistula rat and they did not modify the acid response to G17. Phosphorylation of progastrin-derived peptides is evidently well conserved across a range of species even though there appear to be differences in the relative proportions of phosphorylated and unphosphorylated forms.     

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.     

Selective adsorption/desorption of nucleic acids on submicron-sized polymeric particles

DNA can be removed or separated by the selective adsorption/desorption on positively charged submicronsized polymeric particles (SSPP). The selective adsorption of DNA, in the presence of protein, on positively charged SSPP was accomplished by increasing the concentration of potassium phosphate or sodium phosphate. The adsorption of DNA was not affected by the concentration of potassium phosphate or sodium phosphate up to 1.2M. On the other hand, the adsoprtion of a protein (bovine serum albumin) was completely impeded by 170mM potassium phosphate. DNA adsorbed on SSPP could be desorbed by increasing the concentration of NaCl or KCl, thus it can be recovered. DNA desorbed from SSPP when the concentration of NaCl or KC was higher than 0.6M. A complete desorption of DNA was achieved at the concentration of NaCl or KCl above 1.2M.     

Cdc2 and the Regulation of Mitosis: Six Interacting Mcs Genes

A cdc2-3w weel-50 double mutant of fission yeast displays a temperature-sensitive lethal phenotype that is associated with gross abnormalities of chromosome segregation and has been termed mitotic catastrophe. In order to identify new genetic elements that might interact with the cdc2 protein kinase in the regulation of mitosis, we have isolated revertants of the lethal double mutant. The suppressor mutations define six mcs genes (mcs: mitotic catastrophe suppressor) that are not allelic to any of the following mitotic control genes: cdc2, wee 1, cdc13, cdc25, suc1 or nim1. Each mcs mutation is recessive with respect to wild-type in its ability to suppress mitotic catastrophe. None confer a lethal phenotype as a single mutant but few of the mutants are expected to be nulls. A diverse range of genetic interactions between the mcs mutants and other mitotic regulators were uncovered, including the following examples. First, mcs2 cdc2w or mcs6 cdc2w double mutants display a cell cycle defect dependent on the specific wee allele of cdc2. Second, both mcs1 cdc25-22 or mcs4 cdc25-22 double mutants are nonconditionally lethal, even at a temperature normally permissive for cdc25-22. Finally, the characteristic suppression of the cdc25 phenotype by a loss-of-function wee1 mutation is reversed in a mcs3 mutant background. The mcs genes define new mitotic elements that might be activators or substrates of the cdc2 protein kinase.     

Lethal presentation of mosaic tetrasomy 12p (Pallister-Killian) syndrome

A lethally malformed neonate with mosaic tetrasomy 12p is presented. This is the third reported case of mosaic tetrasomy 12p to have died in the neonatal period. These three babies have shown a consistent phenotype characterized by dysmorphic facies and large diaphragmatic hernia. Mosaic tetrasomy 12p is usually not detectable from lymphocyte investigation, indicating that chromosome studies from cultured fibroblasts should be undertaken in neonates with multiple malformations which include a diaphragmatic defect.     

Genetic linkage of Beckwith-Wiedemann syndrome to 11p15.

Beckwith-Wiedemann syndrome (BWS), characterized by multiorgan developmental abnormalities and predisposition to cancer, usually occurs sporadically, but small apparently dominant pedigrees have been described. Since rare patients show varying karyotypic abnormalities on the short arm of chromosome 11, it has been suggested that BWS may be related to the Wilms tumor gene on 11p13 or, alternatively, to growth factor genes on 11p15. We performed genetic linkage analysis on two BWS kindreds, using RFLPs for loci on 11p. BWS was linked to the insulin gene (11p15.5), with an overall maximum lod score of 3.60 (recombination fraction = .00). Linkage to D11S16 (11p13) could be excluded for recombination fractions less than or equal to .03. These results suggest that BWS defines a tumor-predisposition gene on 11p15.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement and perforin

Previous reports have suggested that a 65 kDa membrane protein, termed homologous restriction factor (HRF), in addition to protecting erythrocytes (E) against lysis by homologous complement (C), may also be involved in protecting cytolytic lymphocytes against lysis mediated by a pore-forming protein (PFP/perforin), one of their own lytic mediators. Here, we used HRF-deficient type III E of patients with paroxysmal nocturnal hemoglobinuria (PNH) to study their susceptibility to lysis mediated by homologous C and perforin, and compared it with lysis of HRF-bearing control or PNH type I E. We show that type III E of PNH patients are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E. In addition, all human E (type I or III) tested here are equally susceptible to lysis mediated by either human (homologous) or murine (heterologous) perforin. By immunoblot analysis, we confirm that type III E, in contrast to type I E, were deficient in the 65 kDa HRF. These results support the notion that homologous species restriction is seen in the C- but not in the lymphocyte perforin-system and argue against an active participation of HRF in protecting cells from perforin-mediated lysis.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

A dynamic model for the structure of acyl carrier protein in solution

The determination of solution structures of proteins using two-dimensional NMR data is commonly based on the assumption that the structure can be represented by a single rigid conformer. We present here a procedure whereby this assumption can be relaxed and illustrate its application to acyl carrier protein from Escherichia coli, a small negatively charged protein with no internal disulfide bonds. The methodology rests on a model having two distinct conformers in dynamic equilibrium. Use of this two-state model results in a dramatic improvement in fit to cross-relaxation-derived distance constraints and a substantial lowering of molecular mechanics energies for individual conformers of acyl carrier protein. The two-state model retains the three-helix motif previously identified on the basis of a one-state structure, but substantial motion of loop regions and the C-terminal peptide, as well as partial disruption of the second helix, is suggested to occur. Support for the existence of these motions can be found in amide exchange rate and spin relaxation time data.