Francisco Barja

CHARACTERIZATION OF SUBPOPULATIONS OF LIPOPROTEIN PARTICLES ISOLATED FROM HUMAN CEREBROSPINAL FLUID

The aim of the present study was to define lipoprotein complexes within cerebrospinal fluid (CSF) in terms of their apolipoprotein composition, using fractionation procedures considered optimal for maintaining lipoprotein structural integrity. Five apolipoproteins were identified, namely apolipoproteins A-I, A-IV, D, E and J. These were differentially distributed amongst lipoprotein particles of which three major subpopulations were identified. CSF-LpAI (20.1 +/- 3.8 nm) was enriched in apolipoprotein A-I and contained the major proportion (> 50%) of apolipoproteins D, E and J. CSF-LpE, of similar size to CSF-LpAI (20.2 +/- 3.1 nm), was composed principally of apolipoprotein E, with minor quantities of apolipoproteins A-I, A-IV, D and J. Elimination of these particles from cerebrospinal fluid by immunoabsorption revealed a third subpopulation of significantly greater diameter (32.0 +/- 6.8 nm). The majority (62%) of apolipoprotein A-IV was also present in this fraction. The study demonstrates the structural and size heterogeneity of lipoproteins in cerebrospinal fluid. This may reflect the lipid transport processes within the central nervous system.

Sensory innervation of the rat portal vein and the hepatic artery

Several neuroanatomical and neurophysiological experiments suggest that the hepatic portal vein is not only richly innervated with sympathetic efferents, but also it is an important source of afferent information. By combining retrograde tracing (with True Blue as a marker) and immunological techniques, the cell bodies for the substance P-containing nerves that surround the portal vein and the hepatic artery of the rat have been localized to the spinal sensory ganglia (T8-T13). Since dorsal root rhizotomy abolished all substance P immunoreactive material from nerve fibres that surround these blood vessels, and since no double-labelled cells were detected in the nodose ganglia, an exclusive spinal origin for the substance P-containing sensory nerves is suggested.

Adrenergic and Peptidergic (Substance P and Vasoactive Intestinal Polypeptide) Innervation of the Rat Portal Vein

The adrenergic innervation of the rat portal vein is subdivided into two plexuses: the adventitial and the medial. Nerve fibres containing immunoreactive substance P (SP) are present in the adventitial and medial plexuses, and extend into the longitudinal and circular muscle layers. Vasoactive intestinal polypeptide (VIP) immunofluorescence is restricted to the adventitial plexus. The catecholamine modulation of the intrinsic rhythmical contractions of the portal vein may be complemented by SP released from intramural nerves and VIP released from the adventitial nerves.

Spinal substance P transmits bradykinin but not osmotic stimuli from hepatic portal vein to hypothalamus in rat

Peripheral osmo - and bradykinin-sensitive receptors which have been previously localised within the hepatic portal vein area, activate the hypothalamo-neuro-hypophysial system through a neural pathway projecting to the lower thoracic spinal cord. In this paper we attempted to identify the spinal transmitter(s) involved and to answer the question whether osmoreceptors are in fact chemosensitive nociceptors. The portal vein of anesthetised rats was superfused with 0.2 ml of 4% NaCl or 1 microM bradykinin, and hypothalamo-neurohypophysial responses were measured either electrophysiologically or by radioimmunoassay of arginine vasopressin. Responses to bradykinin, but not to hypertonic saline, were abolished in rats pretreated 2 wks previously with capsaicin s.c., and immunocytochemistry for substance P in these animals showed that substance P was strongly depleted both in the dorsal thoracic spinal cord and in the portal vein. The spinal injection of 8 microliter 0.1 mM capsaicin at T8-T9 elicited a pronounced hypothalamo-neurohypophysial response, and diminished reversibly the response to bradykinin superfusion of the portal vein. Spinal capsaicin had no effect on responses to hypertonic saline. Similarly, the spinal (T8-T9) injection of 8 micrograms substance P antagonist, the [D-Pro4, D- Trp7 ,9,10, Val8 ]substance P (4-11), reduced reversibly the responses to bradykinin by about 50% without affecting those to hypertonic saline. The spinal injection of 8 micrograms substance P, at the same site where substance P antagonist was applied, elicited within 4 s a prolonged response (several min). A slightly longer delay between stimulus and neurophysiological response was observed for spinal capsaicin and for bradykinin superfusion. Responses to hypertonic saline superfusion of the portal vein, however, occurred within 1-2 s. The results show that portal vein osmoreceptors are distinct from chemo-sensitive nociceptors, and suggest that substance P may be a spinal mediator for chemo-sensitive portal vein nociceptors. The spinal transmitter for osmosensitive afferents, and the physiological importance of the portal vein area in chemosensation remain to be established.

Neural actions of several substance P antagonists in the rat spinal cord.

Four substance P (SP) antagonists were tested on anaesthetized rats, by injecting 8 microgram amounts into the spinal cord (T8-T9), and by observing their effects on the hypothalamo-neurohypophysial responses to a presumably painful stimulus, the superfusion of the hepatic portal vein with 1 microM bradykinin. Only two antagonists, the new D-Pro4,D-Trp7,9,10,Val8-SP4-11 and D-Pro4,D-Trp7,9,10-SP4-11 were capable of inhibiting the responses by 50-60%, the former compound having 3 times less agonistic activity. The results suggest that substitution of the aromatic phenylalanine by the non-polar valine in position 8 may significantly improve the overall characteristics of neurally active SP antagonists.

Zur (FurB) is a key factor in the control of the oxidative stress response in Anabaena sp. PCC 7120

Iron and zinc are necessary nutrients whose homeostasis is tightly controlled by members of the ferric uptake regulator (FUR) superfamily in the cyanobacterium Anabaena sp. PCC7120. Although the link between iron metabolism and oxidative stress management is well documented, little is known about the connection between zinc homeostasis and the oxidative stress response in cyanobacteria. Zinc homeostasis in Anabaena is controlled by Zur, also named FurB. When overexpressed in Escherichia coli, Zur (FurB) improved cell survival during oxidative stress. In order to investigate the possible correlation between Zur and the oxidative stress response in Anabaena, zur deletion and zur-overexpressing strains have been constructed, and the consequences of Zur imbalance evaluated. The lack of Zur increased sensitivity to hydrogen peroxide (H2 O2 ), whereas an excess of Zur enhanced oxidative stress resistance. Both mutants displayed pleiotropic phenotypes, including alterations on the filament surfaces observable by scanning electron microscopy, reduced content of endogenous H2 O2 and altered expression of sodA, catalases and several peroxiredoxins. Transcriptional and biochemical analyses unveiled that the appropriate level of Zur is required for proper control of the oxidative stress response and allowed us to identify major antioxidant enzymes as novel members of the Zur regulon.

Identification and localization of calcium-dependent protease II inNeurospora crassa andUromyces appendiculatus

SummaryThe existence of Ca2+-dependent protease II in crude extracts ofNeurospora crassa andUromyces appendiculatus was demonstrated by immunoblotting using specific antibodies. In both extracts two immunoreacting bands were observed. The molecular mass of the major band inN. crassa corresponded to 37 kDa, while that inU. appendiculatus was 43 kDa, similar to that previously reported forAllomyces arbuscula. Immunofluorescence of the enzyme was predominantly localized in the apical regions of germlings and growing hyphae, suggesting a functional role for the enzyme in hyphal growth.

Nuclear actin and RNA export in conidial cells of Neurospora crassa

We have recently reponed the cytochalasin Bsensitive actin-mediated nuclear RNA export in germinating conidia of Neurospora crassa (Barja and Turian, 1994). We were then led to assume the still undocumented intranuclear presence of actin in nuclei of fungi except slime molds (ref. in LeStourgeon, 1978) contrarily to the long list of references about animal cells (see Milankov and De Boni, 1993). We now have obtained a first, but indirect evidence of it by the radioautographic localization of tritiated cytochalasin B, a technique based on the well-known capacity of this mycotoxin to bind to actin thereby inhibiting its polymerization (Tannenbaum, 1978; Sampath and Pollard, 1991). First to use this technique, Tannenbaum et al. (1973) with [3H]CD, Mayhew et al. (1974) with [3H]CB, showed a localization of Ag grains, and therefore of actin, in animal cells. Direct evidence of nuclear actin in Neurospora, preliminarily obtained by immunoblotting gel separation of extracts from nuclei isolated from normal as well as cytochalasintreated cells, confirms our radioautographies.

Cytochalasin B-sensitive actin-mediated nuclear RNA export in germinating conidia of Neurospora crassa

The mechanism of nuclear RNA export remains elusive (Darnell et aI., 1990; Nigg et aI., 1991; Izaurralde and Mattaj, 1992) in spite of many studies involving several proteins (Borer et al., 1989) including actin (LeStourgeon, 1978). The likelihood that actin can play this role is supported by the fact that it is able to translocate across the nuclear pore complex (Sanger et al. 1980) and that nuclear actin holds nascent RNAs (Nakayasu and Ueda, 1985). Such a contractile protein could serve as the transporter of ribosomal precursors such as preribosomal particles from various regions of the nucleus or nucleolus to the cytoplasm (Bremer et al., 1981). It has also been proposed that myosin participates in such nuclear pore-mediated transport (Schindler and Jiang, 1986).