Subunit structure and function of porcine factor Xa-activated factor VIII

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.     

Time of peak drug concentration after a single dose and a dose at steady state

The time of peak concentration after administration of oral drug is an often quoted and used pharmacokinetic parameter. It is not well appreciated, however, that the peak times after a single dose and a dose at steady state during a multiple administration regimen can differ significantly. This article derives the mathematical relationships that determine how a peak time at steady state differs from that after a single or first dose. These relationships are then evaluated using three different approaches: 1) graphic simulations of time courses of drug concentration for three hypothetical drugs; 2) comparisons of predicted and observed peak times using examples from the literature; and 3) comparisons of predicted and simulated peak times based on different sampling schedules for three hypothetical drugs. The key finding is that peak times after a dose at steady state can occur considerably earlier after administration than after a single dose. However, the manner by which peak times are usually determined, that is, the sampling time corresponding to the highest measured drug concentration, imposes significant limitations on the usefulness of this parameter.     

Activation of auditory cortex during silent lipreading

Watching a speaker's lips during face-to-face conversation (lipreading) markedly improves speech perception, particularly in noisy conditions. With functional magnetic resonance imaging it was found that these linguistic visual cues are sufficient to activate auditory cortex in normal hearing individuals in the absence of auditory speech sounds. Two further experiments suggest that these auditory cortical areas are not engaged when an individual is viewing nonlinguistic facial movements but appear to be activated by silent meaningless speechlike movements (pseudospeech). This supports psycholinguistic evidence that seen speech influences the perception of heard speech at a prelexical stage.     

Changing activation sequence in the embryonic chick heart. Implications for the development of the His-Purkinje system

Viruses that establish persistent infections in their host, such as herpesviruses, adenoviruses or HIV, express proteins designed to pre-empt or evade recognition and elimination by MHC class I restricted CD8+ T lymphocytes. Notable discoveries during the annual period of review have demonstrated that, in principle, each single step within the MHC class I pathway of antigen processing and presentation is fair game for manipulation by viral functions. The viral factors that are natural inhibitors of this pathway have been instrumental for the elucidation of the distinct molecular mechanisms that are exploited by viruses. The viral stealth strategies that downregulate MHC class I protein surface expression may lead, however, to a higher susceptibility of virus-infected cells to natural killer cell activity. Strikingly, there is evidence that some viruses counteract increased natural killer cell recognition by expressing viral MHC class I homologues that function as surrogate inhibitors of natural killer cell activity.     

Phase II study of combined levamisole with recombinant interleukin-2 in patients with advanced malignant melanoma

Adoptive immunotherapy (AI) with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) is an antineoplastic modality in which immune-activated cells are administered to a host with advanced cancer in an attempt to mediate tumor regression. Levamisole (LEV), an immune stimulant, has been suggested to have therapeutic effectiveness in a variety of cancers. After a phase I trial of recombinant IL-2 plus LEV, a phase II trial of this combination was conducted in patients with advanced malignant melanoma. Nineteen patients were entered in the trial. They received IL-2 at 3 x 10(6) U/m2 subcutaneously daily x 5 plus LEV 50 mg/ m2 orally three times daily (p.o. t.i.d.) x 5. Patients were reevaluated at four-week intervals. None of the patients achieved a partial or complete regression (PR, CR). The median time to treatment failure (refusal, progression, or off study due to toxicity) was 56 days. Grade IV toxicities included vomiting (3 patients), lethargy (1 patient), and musculoskellar pain (1 patient). This regimen is not recommended for further testing in patients with advanced malignant melanoma.     

A fetal sheep liver extract reverses age-related increments in spontaneous and induced cytokine production by indirect environmental effects

BALB/c, DBA/2 and C57BL/6 mice of different ages (ranging from 8 to 110 weeks of age) were used as spleen cell donors to assay cytokine production from ConA activated spleen and Peyer's Patch (PP) lymphocytes. As reported in an earlier publication, there was an age-related decline in IL-2 production in all strains, with a general increase in IL-4 and IL-10 production with age, this being particularly marked for PP cell preparations. Similar conclusions were reached from independent analysis of CD44hi and CD44lo cell populations in these groups (memory vs. naive cells, respectively). Interestingly, IL-6 production was dramatically increased (some 4-5-fold in the different strains) and significantly increased levels of IL-6 were detected in the serum of aged mice. A previously described sheep fetal liver extract was able to reverse, to varying degrees, these cytokine changes associated with aging. Interestingly, when cells from aged mice were adoptively transferred to lethally irradiated young (8 week) recipients, the cytokine production phenotype of cells harvested from recipient mice 3 weeks later was that of the aged donor, unless recipients were treated continually with extract. Treatment of the donor alone produced minimal changes in cytokine production 3 weeks following adoptive transfer. The effect of extract was reversed in treated aged mice by concomitant daily intravenous infusion of the competitive inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine (NMMA)), which also decreased the increased serum nitrate levels in mice treated with extract. Our data suggest an important role for reactive nitrogen products, themselves induced by fetal liver extract, in age-associated changes in cytokine production.     

Vegetational association of host-seeking adult blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), on dairy farms in northwestern Wisconsin

Non-obese diabetic NOD/SCID mice have been used to grow human leukaemia as a systemic disease. The animals were inoculated with leukaemic cells obtained from a 36-year-old male with early B-cell precursor acute lymphoblastic leukaemia and on day 15 were given the first of three weekly injections of 1 mg/kg vincristine or equimolar liposomal vincristine. The development of leukaemia in the mice was monitored by taking weekly blood samples and measuring the cell content by flow cytometry. The median time to 50% human cells in the peripheral blood of mice treated with free vincristine was 41 d from the start of treatment compared with 49 d for mice treated with liposomal vincristine (P < 0.01). The median day of death for mice treated with free vincristine was 47 d from the start of treatment and 57 d for mice receiving liposomal vincristine (P<0.01), thus providing a 21% increase in lifespan for animals treated with the liposomal preparation. There was slightly greater weight loss in mice treated with free vincristine than those given liposomal vincristine. Measurement of in vitro colony forming bone marrow progenitor cells in similarly treated, tumour-free mice, showed no difference in progenitor cell survival between mice that received either type of vincristine. We conclude that encapsulating vincristine in liposomes improves the therapeutic index of this drug measured in mice bearing human leukaemia. This may lead to use of the drug in conventional combination chemotherapy with greater safety or, in this setting, at higher dosage.