Cross-linked hemoglobin converts endotoxically inactive pentaacyl endotoxins into a physiologically active conformation

The interaction of purified alpha alpha cross-linked hemoglobin (alpha alpha Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-alpha) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of alpha alpha Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25 degrees to more than 50 degrees. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of alpha alpha Hb interaction. Thus, in the absence of alpha alpha Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of alpha alpha Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to alpha alphaHb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of alpha alpha Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.     

Sertoli-Leydig cell tumor - a rare androgen secreting ovarian tumor in postmenopausal women. Case report and review of literature

Sertoli-Leydig cell tumors (SLCT) constitute only 1-0.5% of all primary ovarian neoplasms. We report a SLCT in a postmenopausal woman aged 69 years. The physical examination revealed severe hirsutism. Basal hormonal evaluation showed high plasma testosterone and estradiol values, with suppressed plasma gonadotropins. Computer tomograph scan revealed a right ovarian tumor mass of 4,3/3 cm, confirming an androgen secreting ovarian tumor. The histopathological and immunocytochemical examination established the diagnosis of well differentiated Sertoli-Leydig cell tumor. The tumor was positive for cytokeratin KL 1 and S-100 protein and, in isolated tumor cells, positive for alpha-fetoprotein. Postsurgical evolution was favorable; controls after 6 months and 3,5 years showed marked reduction of hirsutism, normal plasma testosterone values and gonadotropins in normal postmenopausal range. We discuss the complex aspects of etiology and pathogenesis, the clinical and hormonal settings, the role of immunocytochemical markers in diagnosis, as well as the therapy and the prognostic features of this ovarian tumor.     

Trace metals in sea water. : C. S. Wong,Edward Boyle,Kenneth W. Bruland,J. D. Burton and Edward D. Goldberg. Nato Conference Series,Series IV: Marine Sciences,Volume 9, Plenum Press,New York and London,pp. xiv + 920, ISBN 0-306-41165-2, U.S. $ 115.00.

A comprehensive mechanism is proposed for the photolysis of transition metal complexes in equilibrium solutions. This involves the photo-oxidation, a possibility of energy transfer and several secondary thermal reactions, e.g. forward and back reactions with photochemically-formed H· radicals. The scheme includes the individual quantum yield (IQY) of each species. The photo-oxidations of FeSO₄, FeClO₄, FeCl₂ in aqueous acidic solutions were studied by continuous irradiation. The IQY of Fe_aq²⁺ (0.2), FeSo₄ (0.3), FeHSO₄⁺ (0.67), FeClO₄⁺ (0.06–0.2), FeCl⁺ (0.8), FeCl₂ (0.3) were determined. The IQY was found to be dependent on H⁺ concentration, due to competition with geminal recombination within the solvent cage. This dependence, and the efficiency of energy transfer between excited iron(III) species and iron(II) in ground state, are discussed.     

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h post-reperfusion to distinguish invading monocytes (CD45high/CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-beta and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with cell-type specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia.     

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.     

Syntaxonomy and biogeography of dry grasslands on calcareous substrates in the central and southern Balkans

Questions

Which major syntaxa of dry grasslands supported by carbonate bedrock occur in the central and southern Balkans? What is their position along major ecological gradients and in the context of phytogeographic patterns of the region?

Location

Central and southern Balkans, including western Bulgaria, northern Greece, Kosovo, Macedonia (FYROM) and Serbia.

Methods

We compiled a matrix of 660 relevés of dry grasslands over lime‐rich bedrock, previously classified in the Festuco‐Brometea. We applied clustering techniques to classify separately synoptic and relevé data, and applied NMDS with passive projection of indicator values, climatic data and biogeographic geo‐elements onto ordination diagrams to assist interpretation of the syntaxonomic patterns. We constructed elevation distribution profiles for alliances and classes of grasslands of several grassland classes from a broader study area to elucidate the relationship of the elevational sorting of the syntaxa in relation to latitude.

Results

The analysis revealed six major vegetation types, classified into four orders: (1) Stipo pulcherrimae‐Festucetalia pallentis, incl. (sub)montane rocky steppic grasslands of the Saturejion montanae of central Balkans, and the Koelerio‐Festucion dalmaticae – submontane rocky grasslands of southern Serbia and Kosovo; (2) Astragalo onobrychidis‐Potentilletalia represented by the Saturejo‐Thymion (low‐elevation steppic grasslands of southern Balkans); (3) Festucetalia valesiacae represented by grasslands on deep soil and low elevation of northern Greece, and finally (4) high‐elevation rocky grasslands of southern Balkans, classified as a new alliance – Diantho haematocalycis‐Festucion hirtovaginatae, that might belong to a new, yet undescribed, syntaxonomic order. Ordination suggests that the major differentiation of the high‐rank syntaxa follows north–south geographic and low–high elevation gradients.

Conclusions

Because of the transitional biogeographic position of the studied region, as well as considerable large elevation span across latitudes, the diversity of vegetation types is high. The indication a putative new dry grassland order, the mid‐high altitudes of the southern Balkans points to a need to re‐assess the Balkan vegetation occupying the community niche between the low‐elevation dry grasslands (Festuco‐Brometea) and those typical of high elevations (Elyno‐Seslerietea and Daphno‐Festucetea), seeking parallels to patterns described from the western Alps, Pyrenees, and Apennines. This syntaxonomic unit is poised to expand the concept of the Festuco hystricis‐Ononidetea striatae to the Balkans.

     

System for Measuring Planar Lipid Bilayer Properties

We present a system for measuring planar lipid bilayer properties. The system is composed of a control unit, an output stage, an LCR meter, pumps for filling reservoirs, a bath with temperature regulation and a measurement chamber with four electrodes. The planar lipid bilayer is automatically formed using a folding method on apertures of different sizes. The automatization is assured by two syringes, which are clamped in actuators. Actuators are driven and controlled by a control unit via RS-232 communication. The temperature of the planar lipid bilayer can be regulated between 15 and 55 °C. The regulation is assured by insertion of the measurement chamber into the temperature-regulated bath. Different shapes of voltage- or current-clamp signals can be applied to the planar lipid bilayer. By measuring the response of the planar lipid bilayer to the applied signal, the capacitance and breakdown voltage of the planar lipid bilayer can be determined. The cutoff frequencies of the system output stage for voltage- and current-clamp methods are 11 and 17 kHz, respectively.