Use of triple correlations for the sign determinations of expansion coefficients of symmetric approximations to the diffraction volumes of regular viruses.

An X-ray free electron laser is a new source of x-rays some 10 × 10(9) times brighter than any previous X-ray source, giving rise to the possibility of structure determination of individual biological particles without crystallization. Some of the earliest samples used in the X-ray free electron laser are viruses because they are about the largest of reproducible bioparticles. We show how common virus near-symmetries can be exploited to find a first approximation to their structures to give a starting point for a perturbation approach to determine their structures.

Fiber diffraction without fibers.

Postprocessing of diffraction patterns of completely randomly oriented helical particles, as measured, for example, in so-called "diffract-and-destroy" experiments with an x-ray free electron laser can yield "fiber diffraction" patterns expected of fibrous bundles of the particles. This will allow "single-axis alignment" to be performed computationally, thus obviating the need to do this by experimental means such as forming fibers and laser or flow alignment. The structure of such particles may then be found by either iterative phasing methods or standard methods of fiber diffraction.

Reconstructing an icosahedral virus from single-particle diffraction experiments.

The first experimental data from single-particle scattering experiments from free electron lasers (FELs) are now becoming available. The first such experiments are being performed on relatively large objects such as viruses, which produce relatively low-resolution, low-noise diffraction patterns in so-called "diffract-and-destroy" experiments. We describe a very simple test on the angular correlations of measured diffraction data to determine if the scattering is from an icosahedral particle. If this is confirmed, the efficient algorithm proposed can then combine diffraction data from multiple shots of particles in random unknown orientations to generate a full 3D image of the icosahedral particle. We demonstrate this with a simulation for the satellite tobacco necrosis virus (STNV), the atomic coordinates of whose asymmetric unit is given in Protein Data Bank entry 2BUK.

New light on disordered ensembles: ab initio structure determination of one particle from scattering fluctuations of many copies.

We report on the first experimental ab initio reconstruction of an image of a single particle from fluctuations in the scattering from an ensemble of copies, randomly oriented about an axis. The method is applicable to identical particles frozen in space or time (as by snapshot diffraction from an x-ray free electron laser). These fluctuations enhance information obtainable from an experiment such as conventional small angle x-ray scattering.

Beyond the crystallization paradigm: structure determination from diffraction patterns from ensembles of randomly oriented particles.

We amplify on the principles of the method we have recently proposed for recovering an oversampled diffraction pattern of a single particle from measured diffraction patterns from multiple particles in orientations related by rotation about an axis parallel to the incident radiation. We propose an alternative method of phasing a reference resolution ring by means of a non-negativity constraint on the diffraction intensities, point out the need for caution about enantiomeric ambiguities in the reconstruction of a diffraction pattern from its angular correlations, and show that converged correlations may be deduced by appropriate averaging of even very noisy data.

Prokineticin-1 modulates proliferation and differentiation of enteric neural crest cells.

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

Convergent-beam low energy electron diffraction (CBLEED) and the measurement of surface dipole layers.

We propose the formation of LEED patterns using a highly convergent beam forming a probe of nanometer dimensions. A reflection rocking curve may then be recorded in many diffraction orders simultaneously. Multiple scattering calculations show that the intensity variations within these rocking curves is as sensitive to the parameters describing the surface dipole layer as conventional I/V scans. However the data may be collected from areas sufficiently small to avoid defects and surface steps, radiation damage controlled by use of low voltages, and the information depth selected by choice of the (constant) voltage. We briefly discuss also the application of this method to oxides and the formation of atomic-resolution scanning images in an idealized instrument in which coherent diffracted LEED orders overlap.

Layer spacings in coherently strained epitaxial metal films.

Laterally resolved measurements of the quantum size effect (QSE) in electron reflectivity are made with low energy electron microscopy on coherently strained Ag films on a W(110) surface. The evolution of the total film thickness with increasing number of atomic layers is determined accurately by dynamical theory analysis of the QSE features. Combined with a model of layer spacings obtained from first-principles calculations, this provides for a novel approach to determine the buried interface layer spacing, which is inaccessible to other methods.

An analysis of blood lead data in clinical records by external data on lead pipes and age of household.

This study examined the possibility that lead pipes in the drinking water distribution system were elevating the blood lead levels of children in London, Ontario, Canada. Based on their postal codes, 164 children admitted between 1984 and 1989 to an institution for the behaviorally disordered or developmentally challenged were categorized according to whether they lived in the area of the city known by the local Public Utilities Commission to be serviced by lead pipes. Analysis of covariance was used to obtain confounder-adjusted geometric means in each area. After adjusting for gender, year of lead test (a surrogate for gasoline source), and census tract prevalence of low family income, children in the lead service area (LSA) were found not to have higher blood lead levels (geometric means: LSA = 4.7 micrograms/dl, Non-LSA = 4.8 micrograms/dL; p = 0.839). The average blood lead level declined 60.9% between 1984 and 1989. Using municipal tax assessment data on the age of each child's home, those children living in homes built during or before 1945 (when interior paints were as much as 50% lead by dry weight) had an average blood lead level that was 62.3% higher (p = 0.011) than that of those in homes built since 1975 (when interior paints were limited to no higher than 0.5% lead by dry weight). A clear gradient was observed. This association with age of home remained significant after adjusting for gender, diagnosis, and year of lead test. Variables indicating the amount of industry near the child's residence and the presence of lead service pipes did not enter the model after house-age. In conclusion, no evidence indicated that the lead service pipes were elevating blood lead levels in these London children. The data suggest that with the removal of lead from gasoline, lead-based paint is a significant remaining source of lead exposure. Little data are available on childhood lead exposure from paint in Canada. The present descriptive data suggest that more research into this potential problem in Canada is warranted.

Enzymatic fluorometric assay for plasma pyridoxal 5'-phosphate.

An enzymatic fluorometric assay to quantitate plasma pyridoxal 5'-phosphate (PLP) is described. PLP is preincubated for 30 min with purified tyrosine decarboxylase apoenzyme (TDA) in acetate buffer and is then incubated with L-tyrosine for 60 min. The decarboxylated metabolite, tyramine, is extracted into ethyl acetate, air dried, dissolved in borate buffer and reacted with fluorescamine to form a fluorophor (395/475 nm). Recovery of exogenous PLP was 83%. After log-normal transformation of 129 plasma PLP levels obtained from 3 groups of subjects (in-patients, out-patients, staff), a normal distribution curve was obtained yielding a mean of 83 nmol/L. With 95% confidence level, the reference limits were 32-170 nmol/L. There were no age or sex differences in mean plasma PLP levels between any of the 3 groups of subjects studied.

The lead program at CPRI.

A lead (Pb) screening program in operation at CPRI in London, Ontario, since 1977 involves simultaneous measurement of blood Pb and erythrocyte protoporphyrin (EP) in a randomized population of physically and/or mentally handicapped children and adolescents on admission, discharge and during outpatient visits. This 11-year study has yielded a large database for computerized evaluation. Based upon log normal transformation of data obtained from the admission and outpatient groups, the normal curve yielded a mean and standard deviation (SD) for blood Pb of 0.36 +/- 0.27 mumol/L (n = 4188). This fosters a downward revision of the upper reference limit to 0.89 mumol/L (95% confidence level). The overall mean for EP was 0.35 +/- 0.37 mumol/L and suggests an upper reference limit of 1.09 mumol/L. The direct correlation between annual means of blood Pb and EP retained its significance (r = 0.80; P less than 0.004). For both blood Pb and EP, there was no significant difference in values between admissions (n = 1455), discharges (n = 1310) and outpatient visits (n = 2963). Only in the case of blood Pb was the overall mean value of males (n = 3822) higher (0.46 +/- 0.34 mumol/L) than that of females (0.39 +/- 0.25 mumol/L; n = 1906), but by t-test the difference was not significant. Although annual means of both blood Pb and EP were highest in 1978 and 79 and lowest in 1987, there was no significant difference between any two years.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramuscular administration of iron during long-term chelation therapy with 2,3-dimercaptosuccinic acid in a man with severe lead poisoning.

2,3-Dimercaptosuccinic acid (DMSA) an investigational chelant structurally similar to dimercaptopropanol (BAL), offers the advantage of not depleting iron stores on which basis it would not seem to form a toxic chelate with iron. We report the case of a man with a formidable body burden of lead (Pb) and depleted iron stores who was given iron intramuscularly during a defined period of long-term retreatment with DMSA. Initiation of retreatment with DMSA, 30 mg/kg/day given orally in three divided doses for the first 7 days markedly enhanced Pb diuresis, entailed a pronounced fall in blood Pb and abolished symptoms of Pb poisoning. Continuation of retreatment with two-thirds the initial DMSA dose for an added 15 days maintained blood Pb at sustained low levels. Iron sorbitol administered intramuscularly during this period in individual doses of 100 mg of elemental iron given 3 days apart to a conservative total of 400 mg produced no untoward effects, suggesting that a toxic chelate between iron and DMSA was not formed. Serum ferritin entered the normal range and there was virtually an immediate significant decrease in erythrocyte protoporphyrin. Together with discernible increases in haemoglobin, haematocrit and MCV, this pointed to enhanced iron utilization. Since iron utilization is curtailed by high concentrations of Pb, the immediacy and magnitude of the post-chelation rebound in blood Pb precluded iron administration at any other stage. From these data, DMSA emerges as a uniquely versatile new chelant. Suitable for long-term administration, it permits the simultaneous parenteral administration of iron during dose-related sustained decreases in blood Pb.

Protoporphyrinaemia and decreased activities of 5-aminolevulinic acid dehydrase and uroporphyrinogen I synthetase in erythrocytes of a Vitamin B6-deficient epileptic boy given valproic acid and carbamazepine.

Carbamazepine (CBMZP) has been implicated as an inhibitor of the activities of 5-aminolaevulinic acid dehydratase (ALA-D) and uroporphyrinogen I synthetase (URO-S). In an epileptic boy undergoing long-term treatment with valproic acid (VPA), 1.3 g/d, CBMZP, 0.9 g/d and folic acid, 7.5 mg/d, decreased activities of ALA-D and URO-S coincided with increased levels of erythrocyte protoporphyrin (EP) in the absence of Pb poisoning, iron depletion and erythropoietic protoporphyria. A progressive fall in plasma pyridoxal 5'-phosphate (B6-P) to 7.7 nmol/L (lower reference limit, 14.6 nmol/L) prompted implementation of pyridoxine HCl (B6-HCl), 87.5 mg/d followed by administration of both B6-HCl and preformed B6-P (50 mg/d each). This permitted the eventual withdrawal of VPA and a net reduction of CBMZP to 450 mg/d. During these manipulations, ALA-D and URO-S activities, EP and urinary porphyrins and their precursors were measured serially. An assay system utilizing red cell ALA-D for generation of porphobilinogen (PBG) from added ALA at pH 7.4 was used for determination of ALA-D and URO-S activities in separate aliquots of the same assay mixture both in the absence and presence of Zn and dithiothreitol (DTT). One unit (U) for ALA-D = 1 nmol PBG/L RBC/s; for URO-S = 1 nmol porphyrin/L/s; minimum normal level for ALA-D = 135 U; for URO-S = 6 U. B6-HCl alone entailed increases in ALA-D and URO-S prior to any reduction of CBMZP. After administration of both B6-HCl and B6-P and withdrawal of VPA, the overall increase in ALA-D was from 54.59 to 197.2 U (-Zn; -DTT) and from 50.76 to 217.3 U (+Zn; +DTT). The overall increase in URO-S was from 2.67 to 8.90 U (-Zn; -DTT) and from 3.02 to 8.66 U (+Zn; +DTT). During stepwise reduction of VPA, EP remained elevated to values as high as 2.48 mumol/L (upper reference limit, 1.33 mumol/L). Only after permanent withdrawal of VPA did concentrations of EP fall to normal levels. Values for porphyrins and their precursors in urine were normal throughout. Since both VPA and B6-P are strongly protein-bound, it is suggested that VPA displaced B6-P from protective protein binding sites and that the resulting deficit in B6-P (rather than CBMZP) reduced ALA-D and URO-S activities via primary reduction of ALA-synthetase activity. Increases in EP emerge as a hitherto unappreciated effect of VPA warranting further investigation.

Occupational lead exposure: studies in two brothers showing differential susceptibility to lead.

Unexpected differences in clinical and biochemical findings in two brothers occupationally exposed to the same source of lead for dissimilar lengths of time are presented. Only the brother with the shorter period of lead exposure was anemic and afflicted by nausea, vomiting, abdominal colic and arthralgia. His urinary PBG output yielded the high orders of magnitude found in acute intermittent porphyria in relapse. Prior to administration of a single dose of EDTA (1 g of the calcium disodium salt given intravenously in 325 mL 0.15 mol/L NaCl), his blood lead levels averaged 3.6 mumol/L. The amount of chelatable lead retrieved from his urine, 31 mumol/day, was more than twice that found in his asymptomatic counterpart who was exposed to lead for 13 months and whose pre-EDTA blood lead levels averaged 4.0 mumol/L. Not only the activity of delta-aminolaevulinic acid dehydratase, but also that of uroporphyrinogen I synthetase, was markedly inhibited by lead in red cells of both brothers. These activities were restored to normal levels in vitro by addition to the assay system of zinc and dithiothreitol. This ruled out a coexisting genetic deficiency of either enzyme. The anemia of the symptomatic brother with the shorter period of lead exposure was alleviated by folic acid, 15 mg/day. The differences in findings between the two brothers point to differential susceptibility to lead and illustrate the extent to which symptomatic lead poisoning may mimic biochemical and clinical features of the acute porphyrias.

Opposite changes in blood histamine and force expiratory volume during bronchial inhalation challenge.

Lack of in vivo data on blood histamine changes in subjects during induced attacks of asthma prompted serial determinations of blood histamine levels and of the forced expiratory volume of the first second (FEV1) in 2 asthmatic undergoing antigen (house dust) inhalation challenge and in 3 subjected to methacholine inhalation. In 1 of 2 dust-sensitive asthmatics inhaling house dust (10(4) pnu), a prominent histamine spike occurred 5 minutes after challenge termination, coinciding with a fall in FEV1 by about 15%. In the other, blood histamine had risen steeply at 7 minutes, FEV1 not falling, due presumably to increased bronchial tolerance acquired by hyposensitization therapy. In 1 of 3 asthmatics inhaling methacholine, 0.75 cumulative units/5 breaths (c.u.) entailed a blood histamine spike coinciding with a fall in FEV1 by 26%; after return of both variables to control levels, a second histamine rise preceded a second FEV1 fall. In another, a blood histamine spike coinciding with a fall in FEV1 by 19% occurred 6 1/2 minutes after the methacholine increment affording 99.15 c.u. In the third, FEV1 from the outset fell progressively before blood histamine rose steeply, reaching its lowest value (-26%) likewise with 99.15 c.u. The mechanisms and the source(s) of the histamine accounting for its short-lived increases in blood remain to be determined.