Interaction of a peptidomimetic aminimide inhibitor with elastase.

The crystal structure of an aminimide analog of a dipeptide inhibitor of porcine pancreatic elastase bound to its target serine protease has been solved. The peptidomimetic molecule binds in the same fashion as the class of dipeptides from which it was derived, making similar interactions with the subsites on the elastase surface. Because aminimides are readily synthesized from a wide variety of starting materials, they form the basis for a combinatorial chemistry approach to rational drug design.

Screening for lead poisoning in an urban pediatric clinic using samples obtained by fingerstick.

OBJECTIVE: To assess the false positive rate of blood lead (BPb) determinations on samples obtained by fingerstick from children screened in an urban clinic. METHOD: From a single fingerstick (N = 1573), blood was collected in a capillary tube for determining lead concentration (CPb) by graphite furnace and an additional sample was absorbed onto a filter paper for determining lead concentration (FPb) by atomic absorption spectrophotometry with Delves cup. Zinc protoporphyrin (ZPP) was measured immediately and a confirmatory venous lead (VPb) specimen was obtained at the same visit if the ZPP was > or = 35 micrograms/dL (0.6 mumol/L); children with either a CPb or FPb > or = 15 micrograms/dL (0.7 mumol/L) were later recalled for determining VPb. RESULTS: For the 172 children who had a VPb on the same day as the screening tests, the false positive rates (95% confidence intervals) at a lead threshold of 15 micrograms/dL (0.7 mumol/L) were: CPb, 13.5% (6.7-20.3); FPb, 19.1% (11.8-26.4). Analyses using all 679 screens with a paired venous specimen (mean delay between screen and venous testing = 30 days) yielded much higher false positive rates (CPb, 31.3%; FPb, 46.0%). CONCLUSIONS: Screening for lead poisoning is feasible within an urban pediatric clinic by direct measurement of lead concentration in blood samples obtained by fingerstick. The false positive rate that can be obtained is acceptable given the precision of measuring BPb concentration. Practitioners using a staged screening protocol may incorrectly attribute a higher false positive rate to the screening tests, when much of the error may be due to the temporal variability of BPb resulting from both biologic variability in BPb concentration and intermittent exposures.

In vivo EDRF activity influences platelet function.

The administration of carbachol to rabbits to stimulate the release of endothelium derived relaxing factor (EDRF) results in inhibition of platelet aggregation and elevation of platelet cyclic GMP content. These effects are reversed by simultaneous administration of the EDRF inhibitors methylene blue or haemoglobin. The data provide the first direct biochemical evidence of in vivo EDRF activity.

Atrial natriuretic peptide inhibits the release of endothelium-derived relaxing factor from blood vessels of the rabbit.

We investigated the effects of atrial natriuretic peptide (ANP) on release of endothelium-derived relaxing factor (EDRF) from rabbit aorta and central ear artery using inhibition of platelet aggregation and vascular smooth muscle relaxation respectively as measures of EDRF activity. ANP at concentrations greater than 10(-9) M inhibited carbachol-induced EDRF release from rabbit aorta and acetylcholine-induced EDRF release from the central ear artery. These findings are consistent with previous observations that 8-bromo cyclic GMP inhibits EDRF release from rabbit aorta and ANP inhibits release of EDRF from cultured endothelial cells.

Guillain-Barré syndrome following cardiopulmonary bypass.

Two cases of Guillain-Barré syndrome occurring after otherwise uneventful cardiac surgery using cardiopulmonary bypass are presented. Although Guillain-Barré syndrome has been reported after surgical procedures, it has never been reported after cardiopulmonary bypass. Recent literature supports an immune mediated process for Guillain-Barré. Cardiopulmonary bypass may act as the trigger for this immune mediated response.

Aminimides as peptidomimetics.

Highly selective and specific protein-protein and peptide-protein interactions are elicited by a large number of natural specific biological responses. The mimicry of these natural interactions has been an important and useful strategy for the development of metabolically stable drugs capable of being administered orally (1-4). We report here on the use of a novel type of peptidomimetic based on the aminimide linkage. Aminimides are chemically described as hydrazinium hydroxide inner salts or as ammonium-imines. (Fig. 1) This unusual class of compounds possesses a zwitterionic nature that provides solubility in polar media (water, methanol, ethanol, acetonitrile, DMF, DMSO) as well as in nonpolar solvents such as benzene, when substituents are chosen to reduce the total molecular dipole moment. The aminimide bond has two resonance forms as shown in Fig. 1. ThepKa of the protonated form is generally less than 4. The predominant resonance form will place the formal negative charge on the more electronegative oxygen atom, although we typically use diagrams in which the formal charges reside on the two nitrogens to stress the absence of the hydrogen-bond-donating ability of the aminimide as compared the amide bond in peptides. The aminimide bond is extremely stable under both acidic and basic conditions and is highly resistant to proteolytic cleavage. Fig. 1. Resonance forms for trimethylammoniacetamidate.

The role of verbal estimates of movement error in ballistic skill acquisition.

The role of verbal estimates of movement error in a ballistic movement task was investigated. Two groups performed a rapid linear movement timing task for 50 trials with KR followed by 50 trials without KR. Group I was required to give a verbal estimate of movement time immediately after the task, while Group II was not. Verbal labeling of movement error did not augment performance when KR was present. When KR was withdrawn, however, Group I maintained performance, but Group II's response accuracy declined. Implications for current motor learning theories are discussed.

Directed combinatorial chemistry.

Combinatorial chemistry has given chemists access to vast numbers of molecules, but selecting the right one has proved more difficult. As chemists have gained experience in the technique, however, it has become possible to use solid- or solution-phase syntheses with different chemistries and scaffolds to produce libraries tailor-made for finding or optimizing a lead directed at almost any class of target.

Intramembrane particle distribution and lectin binding of glioblastoma cells after long term subculture.

Human glioblastoma cells in long-term monolayer culture showed an even distribution of intramembrane particles (IMP) on all surfaces of the plasma membrane; junctional complexes were rarely observed and rectilinear arrays were not seen. Cells treated with Con A-ferritin and Ricin II-ferritin showed an even distribution of lectin receptors and under conditions used no capping occurred. Lectin-ferritin complexes were taken up into pinocytotic vesicles. Cleaved preparations of Ricin II-ferritin treated cells showed no change in the distribution of IMP.

Variation in intramembrane components of Trypanosoma brucei from intact and x-irradiated rats: a freeze-cleave study.

Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.

Developing job-related preplacement medical examinations.

Federal regulations prohibiting discrimination in hiring require that employment selection procedures to evaluate applicants be based on job-related criteria. The preplacement physical examination used in employment, particularly in the placement of handicapped persons, must also be conducted in a job-related manner. This paper discusses the development and use of the physical examination in selecting and placing applicants for jobs in the workplace with special reference to handicapped persons and disabled veterans. It presents and justifies a method of performing these examinations in a manner consistent with humanistic and business goals as well as the goals of federal regulatory agencies prohibiting employment discrimination.

The identification and characterization of a STAT 1 binding site in the PPARgamma2 promoter.

Interferon-gamma (IFNgamma) has been shown to decrease the expression of peroxisome proliferator activated receptor-gamma (PPARgamma) in fat cells by blocking the synthesis and increasing the degradation of this transcription factor. Since IFNgamma is a potent activator of STAT 1, we searched for IFNgamma-sensitive binding sites in the PPARgamma promotors. A region of the murine PPARgamma2 promoter was identified that bound nuclear protein from adipocyte nuclei that had been acutely treated with IFNgamma. Supershift analysis revealed that STAT 1, and no other STATs present in the adipocyte nucleus, was capable of binding to this site within the PPARgamma2 promoter. NIH 3T3 and 3T3-L1 cells were transiently transfected with a PPARgamma2 promoter reporter construct, which contained the STAT 1 binding site. Treatment of these cells with IFNgamma resulted in a decrease in reporter activity, demonstrating the modulation of the PPARgamma2 promoter by IFNgamma. We also examined the ability of leukemia inhibitory factor (LIF) to regulate binding at this site. LIF, a potent activator of STAT3 and a weak activator of STAT 1 in these cells, resulted in some binding to the IFNgamma responsive element in the PPARgamma2 promoter that was mediated by STAT 1. Therefore, we examined the ability of LIF to regulate PPARgamma mRNA and observed that LIF, unlike IFNgamma, had little effect on PPARgamma expression. These results and our previous work suggest that cytokine induced STAT 1 homodimers modulate the transcriptional repression of PPARgamma2 in adipocytes.

Intercellular junctions, intramembranous particles and cytoskeletal elements of deep cells of the Fundulus gastrula.

The fine structure of motile deep cells of the gastrula stage of Fundulus heteroclitus was studied with transmission electron microscopy, using both thin sectioning and freeze-cleave techniques. Gastrula deep cells form extensive non-junctiona appositions with each other, in which the apposed plasma membranes are parallel and separated by a distance of 26-28 nm. They also form gap junctions. Tight junctions, desmosomes, and extensive interdigitations of apposed plasma membranes were not observed. The plasma membranes of deep cells contain numerous unclustered intramembranous particles. Cytoplasmic microtubules were found, but they appear to be small in number, sparsely distributed, and mainly randomly oriented. Microfilaments are also present and are localized largely in the cortical cytoplasm and in thin cell extensions. The significance of these findings for the contact and locomotory behavior of deep cells is discussed.

N-acetylcysteine fails to attenuate haemodynamic tolerance to glyceryl trinitrate in healthy volunteers.

1. The effects of chronic dosing with N-acetylcysteine (NAC), on nitrate-induced haemodynamic changes during the acute and chronic treatment of healthy volunteers with glyceryl trinitrate (GTN) patches (Transiderm nitro) has been investigated. 2. Seven volunteers were treated in a double-blind randomised crossover manner for two periods of 4 days with 20 mg of transdermal GTN/24 h together with NAC (200 mg three times daily) or matching placebo. There was a washout period of greater than 3 days between treatment periods. 3. Haemodynamic measurements (blood pressure (BP); heart rate (HR] at rest and following maximal treadmill exercise were performed before treatment and 4 h after starting treatment on days 1 and 4. 4. Significant haemodynamic changes as evidenced by a fall in BP and rise in HR, were seen on day 1 in both the NAC and placebo phases. By day 4 the haemodynamic changes had returned towards the pre-treatment values during both the NAC and placebo phases suggesting the development of tolerance in both treatment groups. 5. These findings suggest that concurrent administration of NAC fails to prevent the development of tolerance to GTN.

Glyceryl trinitrate and platelet aggregation: effects of N-acetyl-cysteine.

The concentration range of GTN causing inhibition of platelet aggregation in vitro is much higher than the plasma concentrations achieved clinically. This action is potentiated by the sulphydryl donor N-acetylcysteine. We have investigated the effects of GTN given with and without N-acetylcysteine on ex vivo platelet aggregation in man. In a double-blind randomised crossover trial eight healthy volunteers were treated with 20 mg of transdermal GTN/24 h, together with N-acetylcysteine 200 mg three times daily or matching placebo. Platelet aggregation, measured ex vivo by whole blood impedance aggregometry in response to adenosine diphosphate, was not significantly altered by GTN acutely or after 4 days' treatment with or without N-acetylcysteine. Platelet cyclic guanosine monophosphate levels were not significantly altered by GTN either in the absence or presence of N-acetylcysteine. This result implies that previously reported beneficial effects of GTN in myocardial infarction or unstable angina are unlikely to be attributable to direct pharmacological inhibition of platelet aggregation.

A production limitation in syllable number: a longitudinal study of one child's early vocabulary.

The present paper reports on the phonological form of one child's productive vocabulary from age 0;10 to 1;8 with primary focus on his production of multisyllabic targets. A large percentage of his multisyllabic vocabulary was produced as one syllable until the age of 1;6. This limitation was not due to a tendency to extract only single syllables from the speech stream, but rather due primarily to a limitation on production. While some portion of his one-syllable productions could be interpreted as the result of single syllable extraction, a sizeable portion affirmed that he extracted the target size correctly by his inclusion of first and final target phonemes in his productions (e.g. [po] for piano and [kiz] for candies). The resolution of this limitation coincides with his move toward two-word speech. We conclude that there is a developmental and perhaps maturational limitation in the capacity to carry out the processes underlying word and sentence production.

Diastolic disease in left ventricular hypertrophy: comparison of M mode and Doppler echocardiography for the assessment of rapid ventricular filling.

OBJECTIVE: To investigate possible discrepancies between M mode and Doppler echocardiography in assessing early diastolic filling. DESIGN: Forty seven patients with left ventricular hypertrophy due to aortic stenosis and 26 healthy controls with a similar age range were studied by M mode, Doppler, apexcardiography, and phonocardiography. The patients also underwent cardiac catheterisation. M mode echograms were digitised by a computer. Early diastolic filling in both groups as assessed by the two techniques was compared. SETTING: A tertiary cardiac referral centre with facilities for non-invasive and invasive investigations. SUBJECTS: Patients referred for assessment of aortic stenosis who had left ventricular hypertrophy. MAIN OUTCOME MEASURES: Filling velocities on Doppler and rates of wall thinning and dimension increase on M mode. RESULTS: Digitised M mode indices of diastolic filling (peak wall thinning rate 6.4 (3.0) v 10.0 (3.0) cm/s and peak rate of dimension increase 9.3 (3.3) v 16 (4.5) cm/s) in the patients and controls were consistently different. In contrast, the Doppler A/E ratio and peak E wave velocity were not; they varied widely among patients with left ventricular hypertrophy. In part, this variability was because the Doppler A/E ratio, but not the digitised M mode indices, was very sensitive to the abnormalities of isovolumic relaxation frequently present in left ventricular hypertrophy. The Doppler A/E ratio varied similarly with age in both normal and hypertrophied hearts; in the patients with ventricular hypertrophy the peak rate of dimension increase depended on age only, whereas the thinning rate was independent of age in both the patients and controls. Neither the A/E ratio nor the M mode indices could be related to the left ventricular end diastolic pressure or the peak aortic pressure difference. CONCLUSIONS: When Doppler and M mode techniques are used to assess rapid filling in patients with left ventricular hypertrophy the M mode indices are more consistently abnormal. The two methods measure different aspects of left ventricular diastolic function and should be regarded as complementary rather than interchangeable.

Chronic administration of N-acetylcysteine fails to prevent nitrate tolerance in patients with stable angina pectoris.

1. Reduced availability of sulphydryl groups in vascular smooth muscle cells may contribute to the development of tolerance to the action of the organic nitrovasodilators. 2. Eight patients with stable angina were treated with 10 mg of transdermal glyceryl trinitrate per 24 h, together with 400 mg N-acetylcysteine, a sulphydryl group donor, or matching placebo three times daily in a double-blind randomised crossover manner for two periods of 4 days with intervening washout period of 3 days off these drugs. Other therapy remained unaltered during the study. 3. Blood pressure, heart rate and symptom-limited treadmill walking time were measured in the pre-treatment control state and 4 h after starting treatment on day 1 and day 4 of each glyceryl trinitrate treatment period. 4. The changes seen on day 1 were attenuated by day 4 to an equal extent in N-acetylcysteine and placebo treatment periods. 5. These results suggest that chronic oral administration of N-acetylcysteine fails to prevent the development of tolerance to the anti-anginal or haemodynamic effects of glyceryl trinitrate.