ABCA3 mutations in adult pulmonary fibrosis patients: a case series and review of literature.

PURPOSE OF REVIEW: The current review aims to recognize the variability in clinical presentation of adult patients with bi-allelic ABCA3 mutations, create more depth in ABCA3 mutations reported and highlight the influence of environmental factors on disease course. RECENT FINDINGS: Mutations in ABCA3 are predominantly linked to neonatal and pediatric interstitial lung disease (ILD) with a minority surviving beyond puberty. Here, we present three patients with ABCA3 mutations who present with disease at the age of 19, 61 and 77. Moreover, we identified c.4451G>C (p.R1484P), c.1675G>A (p.G559R) and c.4745C>G (p.T1582S) as three novel ABCA3 mutations. In addition, we identified six additional patients with ABCA3 mutations in literature who reached an age above 18. Furthermore, we discuss the influence of infections, drugs and smoking on disease course. SUMMARY: Although extremely rare, patients with bi-allelic mutations in ABCA3 may present at adulthood. Late onset of disease may be influenced by type of mutation or environmental factors.

The serum of dysautonomia patients enhances proliferation and signaling in Schwann cells.

Disorders of the autonomic nervous system, or dysautonomias, affect a large segment of the population, especially women, and represent a diagnostic challenge. Identification of biomarkers for autonomic disorders, and the subsequent development of screening methods, would benefit diagnosis and symptom management. We studied the effect of sera from fifteen well-characterized dysautonomia patients (mean age 49+/-16 years, 10 females, 5 males) and ten control subjects (mean age 31+/-14 years, 5 females, 5 males) on the proliferation of cultured Schwann cells and activity of mitogen-activated protein kinases (MAPKs) in these cells. We correlated characteristics of patients with the effects on cell proliferation and signaling. Overall, we observed a significant increase in proliferation when Schwann cells were incubated with sera from female dysautonomia patients when compared to control subjects and male patients. Interestingly, removal of IgGs significantly reduced the proliferative effect of patient sera. We also observed significant activation of p38 MAPK following incubation with both male and female patient sera. These results suggest that patient sera contain factors that contribute to aberrant Schwann cell proliferation and signaling and may ultimately lead to autonomic nerve dysfunction. Our observations represent a promising first step in the identification of dysautonomia biomarkers.

Complications and litigation in gynecologic endoscopy.

Most medical malpractice lawsuits that involve gynecologic endoscopy and laparoscopy result from either improper prevention, inadequate recognition, or delayed intervention. Continuing recognition of this will prevent many and mitigate most such cases. We can learn much from the events and rapid progress of the past decade. Although most laparoscopic improvements have been technical and instrument driven, a basic understanding of anatomy, physiology, and diagnostics remains essential to high-quality patient care and risk reduction.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes. Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline.

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis.

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laevis. The activity of ornithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this development period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage.

Mesoderm Induction in Pleurodeles waltl: Distribution of Fibronectin and Chondroitin Sulfate in Induced Explants: (mesoderm/induction/Pleurodeles/fibronectin/chondroitin sulfate).

In Pleurodeles, cell-matrix interactions play a major role in promoting active mesodermal cell migration during gastrulation. It was therefore important to determine whether the expression of define matrix molecules may be dependent on mesoderm induction. Results from induction experiments done with XTC cell line-conditioned medium show that mesoderm tissues induced in animal cap explants of Pleurodeles are identical to those from Xenopus. However, we also show that dorsally-induced explants in Pleurodeles elongate to a lesser degree than in Xenopus. This observation agrees well with the differences observed in the role of ECM in Pleurodeles and Xenopus gastrulation, respectively. Additional immunostaining studies demonstrate that the induction of mesodermal tissues is associated with the expression of chondroitin sulfate whereas fibronectin fibrils are already assembled in uninduced animal caps. These results suggest that mesoderm cell-matrix interactions in early amphibian embryo may be under the control of mesoderm induction.

Cytochrome P-450 spin state and leakiness of the monooxygenase pathway.

1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (delta alpha) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues. 2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with delta alpha can be experimentally proved. 3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with delta alpha. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O2- which, upon dismutation, gives rise to H2O2. 4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, delta alpha. Concomitantly, the proportion of NADPH accounted for by H2O2 and H2O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties. 5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H2O2 formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H2O2-mediated oxygen tissue toxicity.

The high-spin/low-spin equilibrium in cytochrome P-450--a new method for determination of the high-spin content.

A new method for determination of the population of the high-spin state (high-spin content) in ferric cytochrome P-450 is presented. Based on curve fitting the electronic absorption spectra with a linear combination of gaussian bands analytical functions for the pure high-spin and pure low-spin states were constructed. These functions were used to fit the high-spin/low-spin mixed spectra. A good fit of the absorption spectra of six different cytochrome P-450 proteins in the presence and absence of substrates was found, indicating a similar pi-electron structure of the porphyrin and a similar chemical nature of the nearest coordination sphere of the iron in all cytochrome P-450 proteins.

Demethylation of tertiary amines by a reconstituted cytochrome P-450 enzyme system: kinetics of oxygen consumption and hydrogen peroxide formation.

Initial reaction rates of oxygen consumption and hydrogen peroxide formation in a cytochrome P-450 catalyzed reaction are practically independent of the nature of tertiary amines that were used as substrates. From the kinetic studies and the substrate conversion results that the amount of water formed in a side reaction is determined by the substrate specificity. Both hydrogen peroxide and water formation lower the efficiency of the monooxygenatic activity of cytochrome P-450.

Biosynthesis of sulfated proteoglycans in amphibian embryonal cells.

The synthesis of sulfated proteoglycans in small explants from various parts of late blastulae from Ambystoma mexicanum or Xenopus laevis was investigated by incorporation of radioactive sulfate or glucosamine and galactosamine in media of low, normal or high tonicity. The explants differentiated into ciliated aggregates of fibroblast-like cells, or remained undifferentiated depending upon their origin in the embryo. High tonicity induces the explants to dissociate and prevents morphological differentiation, while low tonicity hardly affects this process. Yet, both types of media decrease the incorporation into glycosaminoglycans to various degrees, ranging from 40 to 80%, depending upon the species. In Xenopus, the uptake of sulfate is inhibited by as much as 90% in high tonicity media. The rate of incorporation of label is approximately twice as much in mesodermal as in animal or vegetal aggregates, which do not differ significantly. Animal aggregates from Ambystoma, however, revealed an exceptionally high uptake of sulfate. The relative distribution of chondroitin sulfates and heparan sulfates is not affected by changes in tonicity, except in Xenopus where high tonicity severely suppresses the synthesis of heparan sulfates, and is independent of the type of aggregate. The relationship between the synthesis of sulfated proteoglycans and processes involved in cell differentiation, especially cell adhesion, is discussed.

Factors involved in the formation and stabilization of cell aggregates obtained from amphibian embryonic explants.

The effect of factors influencing the formation and stability of animal and vegetal aggregates from Xenopus laevis and Ambystoma mexicanum was examined in the light and scanning electron microscopes. At extreme values of pH the surface coat covering the vegetal aggregates is dissolved and dissociation may take place. Animal aggregates are more resistant. At high tonicities vegetal aggregates may be dissociated, and in the animal aggregates the epidermal differentiation is suppressed. In the absence of Ca2+ the vegetal aggregates are dissociated, but the animal aggregates are not affected. The results obtained with the inhibitor selenate and from incorporation experiments indicate that sulfated glycosaminoglycans are involved in the formation of aggregates in both species. Corresponding observations with tunicamycin suggest that even glycoproteins may play a role in aggregate formation, particularly in the vegetal aggregates.

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

p-Nitrophenetole deethylase activity of rat liver microsomes entrapped in polyelectrolyte capsules.

Liver microsomes from phenobarbital induced rats are entrapped in capsules prepared from polyelectrolytes. A comparative analysis of the deethylase activity against p-nitrophenetole by encapsulated and freely suspended microsomes is carried out. The pH optimum occurs at about 7.2 for encapsulated as well as free microsomes. The pH activity profile of encapsulated microsomes, however, is strongly flattened. The maximal velocity of entrapped microsomes is about a quarter of that of free microsomes. The Michaelis-Menten constants are virtually equal. Despite of the lowered activity of encapsulated microsomes this kind of immobilization of enzymes may be useful in biotechnology and medicine because mild immobilization conditions like room temperatures and aqueous solutions are realized.

Changes in activity of the regulatory glycolytic enzymes and of the pyruvate-dehydrogenase complex during the development of Xenopus laevis.

In this study we investigated the variations of the maximal activities of the rate-controlling glycolytic enzymes (i.e., hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK) and of the pyruvate-dehydrogenase complex (PDHc) during the early embryogenesis of Xenopus laevis (from cleavage through hatching). All the enzymatic assays, using different coupled reactions, were performed spectrophotometrically on cytosolic and mitochondrial fractions. The maximal HK activity increases markedly from neurulation onwards, PFK activity presents a peak around gastrulation, PK activity remains relatively constant throughout the period studied and the highest PDHc activity is observed during cleavage. The specific activities display the same temporal pattern. Furthermore, in the sequence of reactions by which glucose is degraded to form acetyl-CoA, the maximal activities of PFK and PK are not limiting while those of HK and PDHc could be rate-limiting at relatively late developmental stages (hatching).

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues.

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.

Relation between the structure of benzphetamine analogues and their binding properties to cytochrome P-450 LM2.

Twelve substrates of a homologous series of tertiary amines (type I substrates) have been reacted with cytochrome P-450 LM2 incorporated into unilamellar liposomes and in soluble form. The apparent spectral dissociation constants (Ks) of the substrate enzyme complexes and the induced high-spin shifts have been correlated with the electron density of distinct carbon atoms as monitored by 13C-NMR chemical shifts, the solubility of the amines and steric parameters of the substrate molecules. The results obtained led to the conclusion that two different intrinsic properties of the substrates can be discriminated in relation to the substrate-enzyme interaction. A diminished electron density at the nitrogen atom is accompanied by an increased binding affinity. The steric structure of the respective substrate determines its capability to shift the spin equilibrium to the high-spin state. Some characteristics of the active center of the enzyme are derived from the evidenced properties of the substrates.

[Regulation mechanisms of the endoplasmic cytochrome P-450 systems of the liver]. / Regulationsmechanismen des endoplasmatischen Zytochrom P-450-Systems der Leber.

Regulation mechanisms of the cytochrome P-450 dependent monooxygenase from the hepatic endoplasmic reticulum at 3 different integrational levels are discussed. At the molecular level the activity of the system is regulated by a substrate dependent shift of an equilibrium of cytochrome P-450 spin conformers in favour of the high spin component. A correlation between the extent of the spin shift, the reduction rate and the substrate turnover has been evidenced. The regulation at the membrane level is based on interactions between the 3 essential components of the system: cytochrome P-450, reductase and lipid. The formation of the cytochrome P-450-reductase-complex necessary for oxygen activation by transfer of electrons is dependent on the charge of the phospholipids. The binding constant of the donor-acceptor complex increases with the acidity of the phospholipid head group, thus enhancing the velocity of the electron transfer.