Increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia

Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15-fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain.     

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.     

Ultrasound-guided injection of contrast medium into the crus penis for diagnosis of erection failure in bulls

The objectives of this study were to demonstrate the ability to cannulate the crurae of the bull's penis under ultrasound guidance, to demonstrate contrast medium injected by this route in the distal penis, and to confirm the technique to be safe and repeatable. Five adult bulls with normal serving ability were used, one being subjected to the procedure twice. The procedure was performed with the bulls under general anesthesia and in lateral recumbency. A spinal needle was passed through the skin and into the crus penis under ultrasound guidance and two syringes containing an iodine-based contrast medium were connected to it. Stimulation using an electro-ejaculator with a rectal probe was initiated, and when the penis started developing an erection, 50-100 ml of contrast medium was injected. Lateral and ventro-dorsal radiographs were taken of the extended penis during, and at intervals after, injection. After a rest period of 5 min, clearance of the contrast medium was confinned and the procedure was repeated on the other crus penis. Each case therefore, contained two attempts. Successful cannulation of the crus penis was confirmed by observing indentation of its fibrous wall by the needle, free flow of blood, lack of resistance to the injection of air, which could be seen in the crus, and fluctuation of resistance to injection in synchrony with the pulsation of the electroejaculator. Contrast medium was demonstrated in the mid or distal portion of the penis in all six cases, or on 9 of the 12 attempts. Attainment of penile erection, a larger volume of contrast medium, and the order of cannulation all enhanced flow of contrast medium to the distal portion of the penis, with the first crus giving better results. On one occasion the needle worked out of the crus penis during stimulation, resulting in injection of contrast medium into the corpus spongiosum penis. All bulls recovered uneventfully and returned to normal serving ability. It is concluded that ultrasound-guided cannulation of the crus penis is a safe and successful method for the injection of contrast medium for contrast studies of the penis, and is less invasive than the surgical method.     

Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP,NADH, and reduced iron

To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from Delta yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from Delta nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation.     

ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.     

Analysis of the catalytic domain of phosphatidylinositol 4-kinase type II

Phosphatidylinositol (PtdIns) 4-kinases catalyze the conversion of PtdIns to PtdIns 4-phosphate, the major precursor of phosphoinositides that regulates a vast array of cellular processes. Based on enzymatic differences, two classes of PtdIns 4-kinase have been distinguished termed Types II and III. Type III kinases, which belong to the phosphatidylinositol (PI) 3/4-kinase family, have been extensively characterized. In contrast, little is known about the Type II enzymes (PI4KIIs), which have been cloned and sequenced very recently. PI4KIIs bear essentially no sequence similarity to other protein or lipid kinases; hence, they represent a novel and distinct branch of the kinase superfamily. Here we define the minimal catalytic domain of a rat PI4KII isoform, PI4KIIalpha, and identify conserved amino acid residues required for catalysis. We further show that the catalytic domain by itself determines targeting of the kinase to membrane rafts. To verify that the PI4KII family extends beyond mammalian sources, we expressed and characterized Drosophila PI4KII and its catalytic domain. Depletion of PI4KII from Drosophila cells resulted in a severe reduction of PtdIns 4-kinase activity, suggesting the in vivo importance of this enzyme.     

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.     

Distinct expression patterns for two Xenopus Bar homeobox genes

The BarH1 and BarH2 homeobox genes are coexpressed in cells of the fly retina and in the central and peripheral nervous systems. The fly Bar genes are required for normal development of the eye and external sensory organs. In Xenopus we have identified two distinct vertebrate Bar-related homeobox genes, XBH1 and XBH2. XBH1 is highly related in sequence and expression pattern to a mammalian gene, MBH1, suggesting that they are orthologues. XBH2 has not previously been identified but is clearly related to the Drosophila Bar genes. During early Xenopus embryogenesis XBH1 and XBH2 are expressed in overlapping regions of the central nervous system. XBH1, but not XBH2, is expressed in the developing retina. By comparing the expression of XBH1 with that of hermes, a marker of differentiated retinal ganglion cells, we show that XBH1 is expressed in retinal ganglion cells during the differentiation process, but is down-regulated as cells become terminally differentiated. Received: 12 August 1999 / Accepted: 5 October 1999