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Studies of the inactivation of factor XIIa by plasma protease inhibitors in purified systems and in plasma were initiated to determine the relative importance of these inhibitors to the neutralization of factor XIIa. Factor XIIa was measured by the amidolysis of H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride or by coagulant activity. C1 inhibitor (C1INH), alpha 2-antiplasmin (alpha 2AP), alpha 2-macroglobulin (alpha 2M), and antithrombin III (ATIII) inhibited factor XIIa with second-order rate constants of 2.2 X 10(5), 1.1 X 10(4), 5.0 X 10(3), and 1.3 X 10(3) M-1 min-1. Factor XIIa activity was not affected by alpha 1-proteinase inhibitor. Incubation of 125I-radiolabeled factor XIIa resulted in 1:1 stoichiometric complexes with C1INH (Mr 190,000), ATIII (Mr 125,000), and alpha 2AP (Mr 150,000 and 125,000) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of 125I-Factor XIIa with alpha 2M resulted in a component of Mr 85,000 on a reduced sodium dodecyl sulfate-polyacrylamide gel, indicating that a subunit of factor XIIa was covalently bound to a proteolyzed portion of alpha 2M. The relative effectiveness of each inhibitor at plasma concentrations was 61:2:3:1 for C1INH, alpha 2AP, alpha 2M, and ATIII, respectively. Kinetic studies of the inactivation of purified factor XIIa added to various plasmas containing different concentrations of C1INH verified the predictions from the purified systems. Gel filtration of radiolabeled factor XIIa incubated with plasma confirmed that factor XIIa-C1INH was the major complex. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complexes in plasma had the same molecular size as those with purified inhibitors. C1INH functions as the predominant inhibitor of factor XIIa in plasma.

The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients with sickle cell anemia or severe thalassemia. Each patient received the drug intravenously for 5 or 7 d. 5-Azacytidine caused a four- to sixfold increase in gamma-messenger RNA concentration in bone marrow cells of eight of the nine patients and decreased the methylation frequency of a specific cytosine residue in the gamma-globin gene promoter in all nine patients. Within 2 d of the start of drug treatment there was a rise in the percentage of reticulocytes containing fetal hemoglobin (HbF; F-reticulocytes) without a significant change in the total number of reticulocytes, which suggested that there was a direct action of 5-azacytidine on erythroid precursors. Late erythroid progenitors (CFU-E), present in bone marrow after 2 d of drug administration, formed colonies containing an increased amount of HbF as compared with control colonies. Moreover, the number of CFU-E derived colonies was not decreased at these early times, which suggested that there was a direct action of 5-azacytidine on erythroid progenitors in the absence of cytotoxicity. Exposure of normal bone marrow cells in tissue culture to 5-azacytidine for 24 h reproduced both of these effects as judged during subsequent colony formation. The combined direct effects of 5-azacytidine on both the erythroid precursor and progenitor compartments resulted in an increase in HbF synthesis that was sustained for 2-3 wk. Toxicity to bone marrow as reflected by cytoreduction was evident after treatment in some patients but was not accompanied by an increase in HbF production. A correlation was found between the effects of 5-azacytidine on bone marrow, as assessed by in vitro measurements, and the hematological response of the individual patients to drug treatment.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.

Earlier Monte Carlo studies on a single-helix model of the GTP cap at the end of a microtubule are extended here to a more realistic five-start helix model of the microtubule end. As in the earlier work, phase changes occur at the microtubule end: the end is either capped with GTP and growing slowly or uncapped and shortening rapidly, and these two regimes alternate (at a given tubulin concentration) at steady state. Macroscopic rate constants for the two-phase model are deduced from the Monte Carlo results. The macroscopic rate constants lead to properties that are in semiquantitative agreement with related experiments of Mitchison and Kirschner.

The recessive hemochromatosis gene is both linked to the HLA region on chromosome 6 and nonrandomly associated with certain HLA alleles. The use of linked HLA markers to trace known hemochromatosis genes within a family is well known, but using the population associations to detect unsuspected disease genes has not been fully appreciated. Thus, while HLA typing has been utilized to detect asymptomatic affected siblings, it has not been applied to other relatives. We propose a method in which Bayes' rule is used to calculate the probability that designated HLA marker haplotypes, brought into the family by spouses, have attendant hemochromatosis genes. The A3, B14 and A3, B7 haplotypes are such high risk markers. When these haplotypes are inherited from the unaffected parent, the offspring of an individual with hemochromatosis is at marked increased risk for the disease. When A3 and B14 are absent from the HLA marker haplotype, however, the risk of having a hemochromatosis gene is less than that for the general population. This approach should be helpful in identifying family members at a higher risk for developing the disease and who may then undergo appropriate periodic screening.

Observations of the course in 20 patients who underwent a long-term anticoagulation due to postthrombotic syndromes resulted in a clinical improvement according to the degrees of severity of the GDR section phlebology as well as in an increase of the plethysmographically measurable venous regurgitation. Thus the late sequelae of the postthrombotic syndrome may possibly reduced by long-term anticoagulation. In one third of the patients despite the anticoagulant therapy a decreased antithrombin-III-value was found.

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex.