The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.     

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

Occurrence of two distinct succinate thiokinases in animal tissues

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Cellular retinoic acid-binding protein and its relationship to the biological activity of four synthetic retinoids in hamster tracheal organ culture

Summary The mechanism of action of retinoid in reversing keratinization in hamster trachea is yet unknown. The purpose of this study was to determine if cellular retinoic acid binding protein (CRABP) is present in tracheal epithelium following incubation in serum-free, vitamin A-deficient culture medium for 10 days, and if the effectiveness of a retinoid in reversing keratinization in organ culture is correlated with its ability to compete for CRABP sites. The cytosol prepared from tracheal cultures contained CRABP at a concentration of 2.61 pmoles per mg protein. Of the four retinoids with carboxyl end group selected for the study, two of the biological active retinoids competed for the CRABP sites. However, no correlation was observed between the biological activity of the inactive retinoids and their ability to associate with the CRABP sites. These results indicate that even though the action of retinoid may be mediated by retinoid binding protein, it cannot be used as a sole predicator of retinoid response in hamster trachea. This investigation was supported by Contract N01-CP-31012 and U. S. P. H. Grants CA30512 and CA32428, which were awarded by the Division of Cancer Etiology, National Cancer Institute, DHHS. Editor's Statement Tracheal organ cultures provide a useful model for the study of epithelial differentiation and carcinogenesis. Much attention has been given to the action of retinoids in this process. Mehta et al. demonstrate a lack of correlation between biological activity and specific cytosolic binding of members of this class of compounds, pointing out the need for a more complete biochemical understanding of the mechanism of action and active forms of retinoids in this and other systems in vivo and in vitro. David W. Barnes     

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific. These results are consistent with the high degree of nonhomology between plasmids R1 and R100 in a 250-bp region covering the distal part of the copB gene and the repA promoter region, which contains the target for the CopB protein.     

The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.