Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.     

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.     

Amyloid precursor protein in the cerebral cortex is rapidly and persistently induced by loss of subcortical innervation

Lesions of the cholinergic nucleus basalis of Meynert elevate the ex vivo synthesis of beta amyloid precursor protein (beta-APP) in the cerebral cortex, a major projection region. We have found that this elevation is reflected by increased levels of beta-APP mRNA. The induction is rapid (occurring 60 min after placement of the lesion) and persistent (remaining for at least 45 days after lesioning). Two other subcortical lesions, which result in reductions of cortical adrenergic and serotonergic innervation, similarly induced cortical beta-APP. The beta-APP induction is reversible and does not require loss of the subcortical neurons. Infusion of lidocaine, a calcium antagonist that disrupts neurotransmitter release, into the nucleus basalis of Meynert leads to the temporary reduction of released acetylcholine in the cortex. In this model, beta-APP mRNA levels are elevated shortly after the infusion of lidocaine (90 min) but return to preinfusion levels 7 days after the lidocaine treatment. However, metabolic stresses of the brain, including chronic physostigmine, glucocorticoid, and diabetogenic treatments, fail to induce the beta-APP response. These results suggest that the induction of beta-APP is a specific response to the loss of functional innervation in the cortex. Importantly, these studies show that cortical beta-APP is induced by lesions that mimic the neurochemical deficits most frequently observed in Alzheimer disease.     

Mifepristone modulation of ACTH and CRH regulation of bovine adrenocorticosteroidogenesis in vitro

In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization. We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig. Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC. The absolute number of plasma cells showed a 10-50-fold increase over the baseline value. Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6. Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis. The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.     

Reduced exploratory activity of audiogenic seizures susceptible Wistar rats

To search for the existence of a tumour-suppressor gene (TSG) associated with oral squamous cell carcinoma (SCC), PCR analysis of microsatellite polymorphisms corresponding to 14 loci which map to chromosome 7q21.3-qter was performed to screen 35 patients with oral SCC for loss of heterozygosity (LOH). LOH was observed in at least one of the loci in 19 of 34 (55.9%) informative cases. Among the loci tested, frequent LOH was restricted at D7S522 on chromosome 7q31.1, which was measured within 1 cM. Furthermore, we detected microsatellite instability (MI) in 11 of 35 (31.4%) cases tested. Our observations indicate that alterations of chromosome 7q are associated with oral SCC tumorigenesis and that 7q31.1 might harbour at least one putative TSG.     

The relationship of blood lead and dietary calcium to blood pressure in the normative aging study

BACKGROUND: Previous studies have demonstrated a positive relationship between elevated blood lead (BPb) and blood pressure (BP), but few have additionally examined the role of dietary calcium. METHODS: The cross-sectional relationship between BPb and BP and the possible protective influence of increased dietary calcium on that relationship was examined among 798 male participants in the Normative Aging Study (NAS), a cohort of older men with relatively low BPb levels. RESULTS: The age range of these subjects was 43-93 years (mean = 66.1, SD = 7.4 years) and blood lead concentrations ranged form 0.5 to 35 mcg/dl (median = 5.6 mcg/dl). For the cohort overall, neither ln blood lead nor dietary calcium were significantly correlated with BP. In multivariate linear regression analyses that adjusted for age, body mass index, dietary calcium intake (adjusted for total calorie intake), alcohol intake, sitting heart rate, kilocalories/week expended in exercise, haematocrit, and smoking status, a unit increase in ln BPb predicted an increase on 1.2 mmHg diastolic blood pressure (DBP) (95% CI : 0.11, 2.2; P = 0.03). Adjusted calcium intake of 800 mg/day predicted a decrease of 3.2 mmHg systolic blood pressure (SBP) (95% CI : -5.6, -0.24, P = 0.03). There was no evidence of an interaction between dietary calcium intake and blood lead on BP. When the analyses were restricted to those men <=74 years old, a unit increase in ln BPb predicted an increase of 1.6 mmHg DBP (n = 681; 95% CI : 0.42, 2.7; P = 0.007). However, when men on antihypertensive medication (AHM) were excluded from the analyses, ln BPb was not significantly associated with increased DBP nor was adjusted calcium significantly associated with SBP. CONCLUSIONS: The study did support the hypothesis that increased BPb was associated with increased DBP in a cohort of older men with low blood lead, but there was no evidence of interaction between BPb and dietary calcium on BP. However, the relationship between increased BPb and DBP did not hold when those on anti-hypertensive medications were excluded.     

Evaluation of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as a new sulfurizing reagent in combination with labile exocyclic amino protecting groups for solid-phase oligonucleotide synthesis

3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) was recently introduced as an efficient sulfurizing reagent for solid-phase oligonucleotide synthesis. The successful syntheses were performed using standard base protecting groups (i.e. benzoyl for A and C, isobutyryl for G), which required deprotection in concentrated ammonium hydroxide at 55 degrees C for 15-18 h. We have explored the possibility of using EDITH in combination with fast deprotection chemistry(e.g. Expedite Chemistry using tert -butylphenoxy acetyl as a base protecting group). Surprisingly, poor synthesis performance was observed when syntheses were conducted with EDITH, Expedite Chemistry and standard synthesis cycle (i.e. Coupling-Thio-Cap). Potential G modification seemed to be the source of incompatibility since sequences containing no G or carrying isobutyryl- protected G residues could be synthesized with high efficiency. However, the deleterious G modification can be readily eliminated by inserting a capping step before the sulfurization reaction. Oligomers prepared with the Coupling-Cap-Thio-Cap cycle contained few phosphodiester contaminants as measured by31P-NMR, anion-exchange HPLC and MALDI-TOF mass spectrometry. In addition to reducing deprotection time, this new combination also provides a mild method for the preparation of certain phosphorothioate oligomers that may be sensitive to prolonged ammonia treatment (e.g. thioated RNAs).     

Vernier and letter acuities for low-pass filtered moving stimuli

Vernier and letter acuities are both susceptible to degradation by image motion. In a previous study, we showed that the worsening of Vernier acuity for stimuli moving up to 4 degrees/s is accounted for primarily by a shift of visual sensitivity to mechanisms of lower spatial frequency. The purposes of this study were to extend the previous results for Vernier acuity to higher stimulus contrast and velocities, and to determine if a shift in spatial scale can similarly explain the degradation of letter acuity for moving stimuli. We measured Vernier discrimination for a pair of vertical abutting thin lines and letter resolution for a four-orientation letter 'T' as a function of stimulus velocity ranging from 0 to 12 degrees/s. Stimuli were presented at 20 times the detection threshold, determined for each velocity. To determine the spatial-frequency mechanism that mediates each task at each velocity, we measured Vernier and letter acuities with low-pass filtered stimuli (cut-off spatial-frequency: 17.1-1.67 c/deg) and analyzed the data using an equivalent blur analysis. Our results show that the empirically determined, equivalent intrinsic blur associated with both tasks increases as a function of stimulus velocity, suggesting corresponding increases in the size of optimally responding mechanisms. This progressive increase in mechanism size can account for the worsening of Vernier and letter acuities with velocity. Vernier discrimination is found to be more susceptible to degradation by various stimulus parameters than letter resolution, suggesting that different mechanisms are involved in the two tasks. We conclude that the elevations in Vernier and letter acuities for moving stimuli are the consequence of a shift of visual sensitivity toward mechanisms of lower spatial frequencies.     

Meckel''s diverticulum: demonstration of heterotopic gastric mucosa with technetium-99m-pertechnetate SPECT

Demonstration of functioning heterotopic gastric mucosa with 99mTc-pertechnetate SPECT is reported. Abnormal tracer uptake was shown conclusively with SPECT but not with planar imaging. When a Meckel's diverticulum is suspected, we suggest SPECT be performed if the results of planar scintigraphy are equivocal and that it be considered if there is a high clinical suspicion and planar imaging is normal.     

Sources of sentence constraint on lexical ambiguity resolution

Results from a series of naming experiments demonstrated that major lexical categories of simple sentences can provide sources of constraint on the interpretation of ambiguous words (homonyms). Manipulation of verb (Experiment 1) or subject noun (Experiment 2) specificity produced contexts that were empirically rated as being strongly biased or ambiguous. Priming was demonstrated for target words related to both senses of a homonym following ambiguous sentences, but only contextually appropriate target words were primed following strongly biased dominant or subordinate sentences. Experiment 3 showed an increase in the magnitude of priming when multiple constraints on activation converged. Experiments 4 and 5 eliminated combinatorial intralexical priming as an alternative explanation. Instead, it was demonstrated that each constraint was influential only insofar as it contributed to the overall semantic representation of the sentence. When the multiple sources of constraint were retained but the sentence-level representation was changed (Experiment 4) or eliminated (Experiment 5), the results of Experiments 1, 2, and 3 and were not replicated. Experiment 6 examined the issue of homonym exposure duration by using an 80-msec stimulus onset asynchrony. The results replicated the previous experiments. The overall evidence indicates that a sentence context can be made strongly and immediately constraining by the inclusion of specific fillers for salient lexical categories. The results are discussed within a constraint-based, context-sensitive model of lexical ambiguity resolution.