Nanostructure of Edge Dislocations in a Smectic-C* Liquid Crystal.

We report on the first direct nanoscale imaging of elementary edge dislocations in a thermotropic smectic-C* liquid crystal with the Burgers vector equal to one smectic layer spacing d. We find two different types of dislocation profiles. In the dislocation of type A, the layers deformations lack mirror symmetry with respect to the plane perpendicular to the Burgers vector; the dislocation core size is on the order of d. In the dislocation of type S, the core is strongly anisotropic, extending along the Burgers vector over distances much larger (by a factor of 4) than d. The difference is attributed to a different orientation of the molecular tilt plane with respect to the dislocation's axis; the asymmetric layers distortions are observed when the molecular tilt plane is perpendicular to the axis and the split S core is observed when the molecules are tilted along the line.

Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor, and lymphotoxin.

Mononuclear cells may be important regulators of fibroblast glycosaminoglycan (GAG) biosynthesis. However, the soluble factors mediating these effects, the importance of intercytokine interactions in this regulation and the mechanisms of these alterations remain poorly understood. We analyzed the effect of recombinant (r) tumor necrosis factor (TNF), lymphotoxin (LT), and gamma, alpha, and beta 1 interferons (INF-gamma, -alpha and -beta 1), alone and in combination, on GAG production by normal human lung fibroblasts. rTNF, rLT, and rINF-gamma each stimulated fibroblast GAG production. In addition, rIFN-gamma synergized with rTNF and rLT to further augment GAG biosynthesis. In contrast, IFN-alpha A, -alpha D, and -beta 1 neither stimulated fibroblast GAG production nor interacted with rTNF or rLT to regulate GAG biosynthesis. The effects of the stimulatory cytokines and cytokine combinations were dose dependent and were abrogated by the respective monoclonal antibodies. In addition, these cytokines did not cause an alteration in the distribution of GAG between the fibroblast cell layer and supernatant. However, the stimulation was at least partially specific for particular GAG moieties with hyaluronic acid biosynthesis being markedly augmented without a comparable increase in the production of sulfated GAGs. Fibroblast prostaglandin production did not mediate these alterations since indomethacin did not decrease the stimulatory effects of the cytokines. In contrast, protein and mRNA synthesis appeared to play a role since the stimulatory effects of the cytokines were abrogated by cyclohexamide and actinomycin D, respectively. In addition, the cytokines and cytokine combinations increased cellular hyaluronate synthetase activity in proportion to their effects on hyaluronic acid suggesting that induction of this enzyme(s) is important in this stimulatory process. These studies demonstrate that IFN-gamma, TNF, and LT are important stimulators of fibroblast GAG biosynthesis, that interactions between these cytokines may be important in this regulatory process, that these cytokines predominantly stimulate hyaluronic acid production and that this effect may be mediated by stimulation of fibroblast hyaluronate synthetase activity.

Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) degradation and human lung fibroblast-derived hyaluronidase.

We characterized the mechanisms by which recombinant (r) tumor necrosis factor (TNF), IFN-gamma, and IL-1, alone and in combination, regulate human lung fibroblast hyaluronic acid (HA) production. Each cytokine stimulated fibroblast HA production. The combination of rTNF and rIFN-gamma resulted in a synergistic increase in the production of high molecular weight HA. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous decrease in HA degradation. In contrast, when rTNF and rIL-1 were combined, an additive increase in low molecular weight HA was noted. This was due to a synergistic increase in hyaluronate synthetase activity and a simultaneous increase in HA degradation. Human lung fibroblasts contained a hyaluronidase that, at pH 3.7, depolymerized high molecular weight HA to 10-40 kD end products of digestion. However, hyaluronidase activity did not correlate with fibroblast HA degradation. Instead, HA degradation correlated with fibroblast-HA binding, which was increased by rIL-1 plus rTNF and decreased by rIFN-gamma plus rTNF. Recombinant IL-1 and rTNF weakly stimulated and rIL-1 and rTNF in combination further augmented the levels of CD44 mRNA in lung fibroblasts. In contrast, rIFN-gamma did not significantly alter the levels of CD44 mRNA in unstimulated or rTNF stimulated cells. These studies demonstrate that rIL-1, rTNF, and rIFN-gamma have complex effects on biosynthesis and degradation which alter the quantity and molecular weight of the HA produced by lung fibroblasts. They also show that fibroblast HA degradation is mediated by a previously unrecognized lysosomal-type hyaluronidase whose function may be regulated by altering fibroblast-HA binding. Lastly, they suggest that the CD44 HA receptor may be involved in this process.

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.

The effect of sphingosine and phorbol ester on the signal transduction enzymes and fibronectin release in cell culture.

In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.

Suggested statistical standards for NTT manuscripts: notes from two of your reviewers.

The purpose of a scientific paper in this journal is to persuade the reader of some important or potentially important facts. For a reader to be persuaded, first the manuscript reviewers must be persuaded, and if the manuscript involves statistical reasoning, at least one of those reviewers is likely to be a statistician. This invited article, by two long-time reviewers for Neurotoxicology and Teratology (NTT) who are also contributors of statistical papers, surveys some of the principles that render a manuscript more persuasive or less persuasive in our eyes. These principles are overwhelmingly not statistical but logical. For one typical NTT manuscript theme, the relation between some toxic exposure and one or more negative outcomes in humans, the aspects of manuscripts we scrutinize most closely include biological plausibility, dose-response relationships, breadth of evidence, adjustments for measurement bias, attention to assumptions and scatterplots in the search for confounds, and, in general, a sincere attempt to enunciate and then refute plausible hypotheses rival to the one the investigators prefer. The literature of excellent studies in other fields provides ample instances of good practice in these matters; we review it in those fields for applications in ours. Formal statistical significance testing plays almost no role in the most persuasive papers. In particular, findings that appear only after "adjustment for covariates" are never considered credible by these reviewers; we explain our reasons at length, and suggest alternatives.

Investigation of bioisosteric effects on the interaction of substrates/ inhibitors with the methionyl-tRNA synthetase from Escherichia coli.

Aminoacyl-tRNA synthetases catalyze the stepwise coupling of specific amino acid substrates to their cognate tRNAs. The first intermediate formed in this process is the aminoacyl-adenylate, which then subsequently reacts with the 3'-terminus of the cognate tRNA to transfer the amino acid to the tRNA. This overall reaction is critical for protein biosynthesis and is quintessential to the viability of all organisms. Therefore, the selective inhibition of bacterial amino acid-tRNA synthetases is the focus of intense current interest for the development of novel antibacterial agents. In order to elucidate some of the critical factors involved in recognition and binding of potential inhibitors to these bacterial systems, the current report has focused on the methionyl-tRNA synthetase from Escherichia coli. This enzyme has been studied with two sets of bioisosteric replacements in the methionine and methionyl-adenylate structures. Replacements of the carboxyl group of methionine with the phosphinic and phosphonic acid moieties were used to probe the effects of including potential transition state analogs on enzyme inhibition. The contributions of the aminoacyl-adenylate structure and the effect that fluorination has on inhibitory activity were investigated utilizing 5'-O-[(L-methionyl)-sulfamoyl]adenosine and 5'-O-[(S-trifluoromethyl-L-homocysteinyl)-sulfamoyl]adenosine. The K(i) values for these compounds were determined to be 0.4 mM, 1.2 mM, 0.25 nM and 2.4 nM respectively. A discussion of this data in relation to structural information provided by the recent determination of the three-dimensional structures of the E. coli enzyme with several of these compounds is presented.

Analogues of aminoglutethimide: selective inhibition of aromatase.

In exploring further the structural features that influence the relative efficacy of analogues of aminoglutethimide [1, 3-(4-aminophenyl)-3-ethylpiperidine-2,6-dione] as inhibitors of the cholesterol side-chain cleavage enzyme system desmolase and the estrogen forming system aromatase, analogues have been synthesized in which the aminophenyl substituent is replaced by pyridyl or substituted pyridyl. The 4-pyridyl analogue 5 [3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione] is a strong competitive inhibitor of aromatase (Ki = 1.1 microM; value for 1, 0.60 microM), which exhibits a type II difference spectrum (Ks = 0.28 microM; value for 1, 0.13 microM) but is noninhibitory toward desmolase. The 2- and 3-pyridyl analogues (3 and 4) inhibit neither enzyme system. 1-Amino-3-ethyl-3-phenylpiperidine-2,6-dione (2) is a strong and selective inhibitor of desmolase but the 4-pyridyl analogue 10 [1-amino-3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione] is a weak inhibitor of desmolase and aromatase. Analogues of 5 having a less basic aromatic substituent, namely, the N-oxide 11 and the 2,3,5,6-tetrafluoro derivative 13, were also prepared. The latter is a weak inhibitor of aromatase and the former inhibits neither enzyme system.

Analysis of facial shape in children gestationally exposed to marijuana, alcohol, and/or cocaine.

The association between fetal marijuana and/or alcohol exposure and facial features resembling fetal alcohol syndrome was investigated in a sample of 80 children. Standardized lateral and frontal facial photographs were taken of 40 children, 5 to 7 years of age, whose mothers reported frequent use of marijuana during the first trimester of pregnancy and 40 children whose mothers reported no use of marijuana during pregnancy. The marijuana-exposed and unexposed children were group-matched on alcohol exposure prior to and during pregnancy, sex, race, and age at the time of assessment. The photographs were assessed clinically by a study staff dysmorphologist and morphometrically by computerized landmark analysis. Fetal alcohol syndrome-like facial features were not associated with prenatal marijuana exposure in this study sample. No consistent patterns of facial features were identified among the marijuana-exposed group. Maternal consumption of two or more ounces of alcohol per day, on average, in early gestation was found to be associated with fetal alcohol syndrome-like facial features identified both clinically and morphometrically. Cocaine use reported by 13 of the 80 women was independently associated with mild facial dysmorphic features of hypertelorism and midfacial flattening. The results demonstrate the usefulness of this diagnostic technique for quantifying anomalies apparently unique to fetal alcohol syndrome and for targeting clusters of anomalies in new conditions for future evaluation.

On categorizations in analyses of alcohol teratogenesis.

In biomedical scientific investigations, expositions of findings are conceptually simplest when they comprise comparisons of discrete groups of individuals or involve discrete features or characteristics of individuals. But the descriptive benefits of categorization become outweighed by their limitations in studies involving dose-response relationships, as in many teratogenic and environmental exposure studies. This article addresses a pair of categorization issues concerning the effects of prenatal alcohol exposure that have important public health consequences: the labeling of individuals as fetal alcohol syndrome (FAS) versus fetal alcohol effects (FAE) or alcohol-related neurodevelopmental disorder (ARND), and the categorization of prenatal exposure dose by thresholds. We present data showing that patients with FAS and others with FAE do not have meaningfully different behavioral performance, standardized scores of IQ, arithmetic and adaptive behavior, or secondary disabilities. Similarly overlapping distributions on measures of executive functioning offer a basis for identifying alcohol-affected individuals in a manner that does not simply reflect IQ deficits. At the other end of the teratological continuum, we turn to the reporting of threshold effects in dose-response relationships. Here we illustrate the importance of multivariate analyses using data from the Seattle, Washington, longitudinal prospective study on alcohol and pregnancy. Relationships between many neurobehavioral outcomes and measures of prenatal alcohol exposure are monotone without threshold down to the lowest nonzero levels of exposure, a finding consistent with reports from animal studies. In sum, alcohol effects on the developing human brain appear to be a continuum without threshold when dose and behavioral effects are quantified appropriately.

Facial effects of fetal alcohol exposure: assessment by photographs and morphometric analysis.

This study was designed to assess the limits of alcohol-related facial dysmorphogenesis. Standard full face and lateral facial photographs were obtained on 21 7-year-old children who had been exposed gestationally to known, heavy quantities of ethanol. Only two of these children had been previously considered to have definite fetal alcohol syndrome (FAS). Similar photographs of 21 other 7-year-old children with negligible gestational ethanol exposure were obtained for control purposes. Copies of the 42 photographs were given to each of seven expert clinicians who were asked to select any child with an FAS-related facial appearance. Six of seven judges were accurate in identifying children with high levels of alcohol exposure as having a fetal alcohol-affected face. A set of homologous points on the photographs were then digitized and analyzed by newly developed morphometric methods to determine the facial shape characteristics that distinguish the selected photographs of highly exposed children. The analysis confirmed that the judges specifically identified children with facial changes consistent with those previously published as defining the face of the FAS: short palpebral fissures, a relatively long and flat midface, and a retrusive mandible. This methodology may be useful in more accurately delineating the facial phenotype in other conditions diagnosed primarily on the basis of subjective clinical criteria.

A preliminary statistical examination of the effects of uncertainty and variability on environmental regulatory criteria for ozone.

Basing the quantitative expression of environmental regulatory standards and associated compliance criteria on statistical principles has recently received attention in Europe, most visibly in a study by the UK Royal Commission on Environmental Pollution. These issues are timely for consideration in the USA, where a recent periodic review of National Ambient Air Quality Standards (NAAQS) has led to revision of the regulatory standards for ambient ozone and particulate matter. Salient statistical issues include accounting for errors of the first and second kind due to sampling and measurement error. These issues appear routine statistically and also may seem absent from regulations, but neither is necessarily the case. This paper is directed towards developing a methodology for examining the problem of dealing with uncertainty and variation in environmental regulations and compliance criteria. Our approach is illustrated through statistical analysis of the (old) 1 hour and the (new) 8 hour standards for ambient ozone, based on intensive monitoring in California's San Joaquin Valley during summer 1990 performed under the SARMAP Project. This paper presents preliminary findings based on quantifying measurement error or precision in terms of small-scale spatial and temporal variability, laying the groundwork for future work.

"Size" and "shape" variables in the presence of covariates: an application to the sudden infant death syndrome.

A "size measure independent of shape" is used to investigate differences in "size" and "shape" variables, defined as functions of weight and length at birth, between a random control group and victims of the Sudden Infant Death Syndrome (SIDS). At birth, the SIDS group has a lower mean size than the control group. After adjusting for maternal and environmental factors (covariates), there is a lessened but persistent difference in size, suggesting the existence of some undetected factor linked to sudden death in infants.

Studying alcohol teratogenesis from the perspective of the fetal alcohol syndrome: methodological and statistical issues.

Alcohol is a teratogenic drug and the effects appear to be grossly dose related. The severest effects are observable clinically as the fetal alcohol syndrome and are associated with heavy prenatal alcohol exposure and a history of chronic maternal abuse of alcohol. Hypotheses for subtler behavioral effects associated with lower levels of exposure can be generated from observation of the behavioral effects in FAS. Behavioral effects associated with various levels of prenatal alcohol exposure in humans include poor sucking and poor habituation in the newborn, poorer mental and motor development in infancy, and attentional and reaction time effects at four and seven years of age. Human behavioral teratology studies are necessarily complex due to the large number of covariates that affect behavior, modify the effects of teratogens, and influence interpretation. Challenging problems exist in assessing exposure, outcomes, and covariates. Common to assessment of all these classes of variables are the multiplicity of measurement and the indirect nature of the measurement. Answering specific questions about timing and dose effects demands careful statistical modeling procedures and large, complex data bases. Large data bases and indirect measurement problems suggest factor analytic extensions of current regression methodology, which we have proposed in this paper.

Permeability characteristics of cultured endothelial cell monolayers.

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.

Effect of 100% O2 on passage of uncharged dextrans from blood to lung lymph.

Since charge as well as size may influence the passage of plasma proteins from blood to lung lymph, we used uncharged dextrans as tracers to study the effects of hyperoxic lung injury on the molecular sieving properties of the pulmonary microcirculation in unanesthetized sheep. Polydisperse [3H]dextran was infused intravenously into five sheep before and after the animals breathed 100% O2 until lymph flow increased threefold (66-84 h). Lymph-to-plasma concentration ratios (L/P) were determined for [3H]dextran fractions of graded molecular sizes (1.6-8.4 nm effective radius) from samples obtained during the infusions. Before hyperoxia the blood-lymph barrier was highly restrictive to transport of [3H]dextrans above 5.0 nm in radius; steady-state L/P for these molecules averaged 0.03 or less. After the sheep breathed 100% O2, [3H]dextrans as large as 8.4 nm radius appeared in the lymph. Posthyperoxia, the L/P were significantly increased relative to prehyperoxia base-line values for every [3H]dextran fraction larger than 2.0 nm radius (P less than 0.05). In contrast, neither the L/P for albumin or total protein changed significantly. At autopsy, electron microscopy showed widespread damage to the endothelium of the alveolar capillaries with infrequent gaps between endothelial cells. In two control sheep, inhalation of compressed air for 96 h had no effect on lymph flow or L/P for the [3H]dextrans. We conclude that O2 poisoning reduced the selective sieving of uncharged dextrans across the blood-lymph barrier of the lungs and allowed larger dextrans to enter the lymph. These larger molecules may have leaked from the pulmonary microcirculation via disruptions in the continuity of the endothelial lining.

Passage of uncharged dextrans from blood to lung lymph in awake sheep.

To examine how molecular size alone influences the passage of macromolecules from the pulmonary microcirculation into lymph collected from the caudal mediastinal lymph node of the sheep, we infused polydisperse uncharged [3H]dextrans intravenously at a constant rate over a period of 7.5 h in nine awake sheep with lung lymph fistulas. Lymph and plasma were collected during hours 5.5-7.5 of the infusions, and the [3H]dextrans were separated by molecular sieve chromatography into fractions that ranged from 1.6 to 8.4 nm in effective molecular (Stokes-Einstein) radius. Lymph-to-plasma (L/P) ratios for [3H]dextrans were near 1.0 at 1.6-nm radius, decreased with increasing molecular size, and approached zero at radii above 5.0 nm. We confirmed that these L/P ratios represented steady-state values by extending the duration of the infusion to approximately 30 h in two of the nine sheep and finding that the L/P ratios remained unchanged. These results were consistent with molecular sieving through a homoporous membrane with cylindrical pores of 5.0-nm radius. We also found that the L/P ratio for albumin [0.76 +/- 0.13 (SE)] in five of the same sheep was much higher than that for the [3H]dextran fraction of the same effective molecular radius [0.11 +/- 0.02 (SE)]. These results suggest that the movement of macromolecules from the pulmonary microcirculation into pulmonary lymph collected from the caudal mediastinal node of the sheep is influenced by both molecular size and molecular charge and that, compared with uncharged dextrans, the steady-state passage of anionic endogenous proteins from plasma to lymph is enhanced.

Association of prenatal alcohol exposure with behavioral and learning problems in early adolescence.

OBJECTIVE: To examine the association of moderate levels of prenatal alcohol exposure with learning and behavior in early adolescence. METHOD: A population-based cohort of 464 children were followed longitudinally from birth to age 14 years. Alcohol exposure was assessed via in-depth maternal self-report in the fifth month of pregnancy. At age 14, learning and behavior were assessed with multiple measures, tapping parent, teenager, and psychologist viewpoints, drawn from adolescent laboratory examination and parent phone interview. The underlying pattern of association between prenatal alcohol and adolescent outcome was detected using partial least-squares statistical techniques; confounding factors were dealt with by regression methods. RESULTS: Analyses revealed a statistically significant, subtle relationship between greater prenatal alcohol use and increased behavior/learning difficulties during adolescence, even after accounting for other developmental influences. "Binge" maternal drinking and exposure early in pregnancy were associated with a profile of adolescent antisocial behavior, school problems, and self-perceived learning difficulties. CONCLUSIONS: Fetal alcohol exposure (even at "social drinking" levels) is associated with developmental difficulties in adolescence that are consistent with problems seen earlier in life. Clinicians should understand the potential role prenatal alcohol exposure plays in behavioral and cognitive problems.

Prenatal alcohol and offspring development: the first fourteen years.

This report summarizes findings from a prospective longitudinal study of the effects of prenatal alcohol exposure on a birth cohort of 500 offspring selected from 1,529 consecutive pregnant women in prenatal care by mid-pregnancy at two representative community hospitals. Effects of prenatal alcohol observable on size measures at birth were insignificant after 8 months. Morphometric analysis of facial features identified effects only at the very highest alcohol exposure levels. By contrast, dose-dependent effects on neurobehavioral function from birth to 14 years have been established using partial least squares (PLS) methods jointly analysing multiple measures of both alcohol dose and outcome. Particularly salient effects included problems with attention, speed of information processing, and learning problems, especially arithmetic.