Periphere okklusive Vaskulopathie bei einer Patientin mit kombinierter Präeklampsie und Alpha-Thalassämie minor [Peripheral occlusive retinal vasculopathy in a woman with combined preeclampsia and alpha thalassemia minor].

Normalerweise eine Störung der ersten Schwangerschaft, ist die Präeklampsie charakterisiert durch eine arterielle Hypertonie (> 140 mmHg systolisch oder > 90 mmHg diastolisch), die in der Regel nach der 20. Schwangerschaftswoche auftritt und von einer Proteinurie begleitet wird [1]. Die Präeklampsie wird als ,,schwer" bezeichnet, wenn sie mit einer wesentlichen Erhöhung des Blutdrucks (> 160 mmHg systolisch oder > 110 mmHg diastolisch), schwerer Proteinurie, Oligurie, Lungenödem, abdominalen Schmerzen, Leberfunktionsstörungen, Thrombozytopenie und visuellen oder zerebralen Symptomen einhergeht. Eine Eklampsie wiederum ist durch die Entwicklung von tonisch-klonischen Anfällen bei einer präeklamptischen Patientin charakterisiert. Bei der Alpha-Thalassämie tritt ein Defekt von 2 oder mehr der 4 Alpha-Globin-Gene auf. Von einer Alpha-Thalassämie minor spricht man, wenn 2 Alpha-Ketten-Gene deletiert sind. Sie tritt häufig bei Menschen aus Afrika, Südostasien, dem westindischen und mediterranen Raum auf. Die Alpha-Thalassämie minor verursacht eine milde bis moderate mikrozytäre Anämie. Wir berichten über eine Patientin mit peripherer okklusiver Vaskulopathie im Rahmen einer kombinierten Präeklampsie und Alpha-Thalassämie minor.

Pi*-pi* bonding interactions generated by halogen oxidation of zirconium(IV) redox-active ligand complexes.

The new complex, [Zr(pda)2]n (1, pda2- = N,N'-bis(neo-pentyl)-ortho-phenylenediamide, n = 1 or 2), prepared by the reaction of 2 equiv of pdaLi2 with ZrCl4, reacts rapidly with halogen oxidants to afford the new product ZrX2(disq)2 (3, X = Cl, Br, I; disq- = N,N'-bis(neo-pentyl)-ortho-diiminosemiquinonate) in which each redox-active ligand has been oxidized by one electron. The oxidation products 3a-c have been structurally characterized and display an unusual parallel stacked arrangement of the disq- ligands in the solid state, with a separation of approximately 3 A. Density functional calculations show a bonding-type interaction between the SOMOs of the disq- ligands to form a unique HOMO while the antibonding linear combination forms a unique LUMO. This orbital configuration leads to a closed-shell-singlet ground-state electron configuration (S = 0). Temperature-dependent magnetism measurements indicate a low-lying triplet excited state at approximately 750 cm-1. In solution, 3a-c show strong disq--based absorption bands that are invariant across the halide series. Taken together these spectroscopic measurements provide experimental values for the one- and two-electron energies that characterize the pi-stacked bonding interaction between the two disq- ligands.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Interaction of alpha-melanotropin with central dopamine systems: role of hormonal state and molecular structure

The tubero-infundibular and nigrostriatal DA neurone systems of rats respond to systemic (i.p.) injection of alpha-MSH (2-100 microgram/kg). The response of the tubero-infundibular (arcuate) DA neurones, an increase in cellular fluorescence intensity which can be interpreted as a sign of increased neuronal activity, is essentially the same in males, estrogen-progesterone-pretreated ovariectomized females and hypophysectomized males, whereas the type of response elicited by alpha-MSH in the nigral DA neurones depends upon the hormonal state of the animal. Differences between the two DA neurone groups exist also with regard to the effects of peptide fragments containing the two active sites of the alpha-MSH molecule. Results of lesion experiments in the lower brainstem (area postrema) and of blockade of muscarinic mechanisms by atropine further point to differences in the mechanisms underlying the peptide effects on the two neurone systems. The reaction of the tubero-infundibular DA system (which controls the pars intermedia of the pituitary) can be considered to reflect the activation of a feedback mechanism on MSH secretion, while the functional counterpart of the changes observed in the nigral system remains unknown at the present time.

A pilot assessment of alpha-stat vs pH-stat arterial blood gas analysis after cardiac arrest.

PURPOSE: Resuscitated cardiac arrest (CA) patients typically receive therapeutic hypothermia, but arterial blood gases (ABGs) are often assessed after adjustment to 37°C (alpha-stat) instead of actual body temperature (pH-stat). We sought to compare alpha-stat and pH-stat assessment of Pao2 and Paco2 in such patients. MATERIALS AND METHODS: Using ABG data obtained during the first 24 hours of intensive care unit admission, we determined the impact of measured alpha vs calculated pH-stat on Pao2 and Paco2 on patient classification and outcomes for CA patients. RESULTS: We assessed 1013 ABGs from 120 CA patients with a median age of patients 66 years (interquartile range, 50-76). Median alpha-stat Pao2 changed from 122 (95-156) to 107 (82-143) mm Hg with pH-stat and median Paco2 from 39 (34-46) to 35 (30-41) mm Hg (both P < .001). Using the categories of hyperoxemia, normoxemia, and hypoxemia, pH-stat estimation of Pao2 reclassified approximately 20% of patients. Using the categories of hypercapnia, normocapnia, and hypocapnia, pH stat estimation of Paco2 reclassified approximately 40% of patients. The mortality of patients in different Pao2 and Paco2 categories was similar for pH-stat and alpha-stat. CONCLUSIONS: Using the pH-stat method, fewer resuscitated CA patients admitted to intensive care unit were classified as hyperoxemic or hypercapnic compared with alpha-stat. These findings suggest an impact of ABG assessment methodology on Pao2, Paco2, and patient classification but not on associated outcomes.

A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

TCR-alpha CDR3 loop audition regulates positive selection.

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-beta-transgenic mice in which MHC shapes TCR-alpha use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of alphabeta pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the alpha CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.

EEG alpha activity reflects motor preparation rather than the mode of action selection

Alpha-band activity (8-13 Hz) is not only suppressed by sensory stimulation and movements, but also modulated by attention, working memory and mental tasks, and could be sensitive to higher motor control functions. The aim of the present study was to examine alpha oscillatory activity during the preparation of simple left or right finger movements, contrasting the external and internal mode of action selection. Three preparation conditions were examined using a precueing paradigm with S1 as the preparatory and S2 as the imperative cue: Full, laterality instructed by S1; Free, laterality freely selected and None, laterality instructed by S2. Time-frequency (TF) analysis was performed in the alpha frequency range during the S1-S2 interval, and alpha motor-related amplitude asymmetries (MRAA) were also calculated. The significant MRAA during the Full and Free conditions indicated effective external and internal motor response preparation. In the absence of specific motor preparation (None), a posterior alpha event-related desynchronization (ERD) dominated, reflecting the main engagement of attentional resources. In Full and Free motor preparation, posterior alpha ERD was accompanied by a midparietal alpha event-related synchronization (ERS), suggesting a concomitant inhibition of task-irrelevant visual activity. In both Full and Free motor preparation, analysis of alpha power according to MRAA amplitude revealed two types of functional activation patterns: (1) a motor alpha pattern, with predominantly midparietal alpha ERS and large MRAA corresponding to lateralized motor activation/visual inhibition and (2) an attentional alpha pattern, with dominating right posterior alpha ERD and small MRAA reflecting visuospatial attention. The present results suggest that alpha oscillatory patterns do not resolve the selection mode of action, but rather distinguish separate functional strategies of motor preparation.

The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.

Mutational and computational analysis of the alpha(1b)-adrenergic receptor. Involvement of basic and hydrophobic residues in receptor activation and G protein coupling.

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. &amp; Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

Rapid identification of malaria vaccine candidates based on alpha-helical coiled coil protein motif.

To identify malaria antigens for vaccine development, we selected alpha-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally "native" epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high alpha-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.