Bodenin: a novel murine gene expressed in restricted areas of the brain

Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the alpha1-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: Jv(c,f)=262+/-54 compared with 350+/-17 mm-2, control compared with prazosin (P<0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5+/-0.9% after 2 weeks (P<0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7+/-0. 8% of controls; 7.1+/-1.9% at 1 week; 8.8+/-2.5% at 2 weeks; P<0.02), some of which were smaller in size (less than 2 microm diameter) than in controls (3-5 microm) and/or "seamless", i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5+/-6.2%, P<0.001) and 2 weeks of prazosin (34.2+/-5.5%, P<0.05) than in control vessels (16.7+/-3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks' prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.     

Neurosteroids. Neuromodulators of cerebral excitability

Recent advances in the understanding of the pathogenesis of insulin-dependent diabetes mellitus (IDDM) have led to the first trials of disease prevention in susceptible individuals. Two main trials (nicotinamide and insulin) are now running but first results will not be available before the turn of the century. Pilot trials using different approaches, most of them based on the induction of immunotolerance, are also under way and should offer new insight for establishing larger multicentre studies including attempts aimed at primary prevention by removal of diabetogenic components in cow's milk. The field is moving fast and it is expected that intervention for IDDM prevention will be offered to an increasing number of individuals found at risk of developing the disease.     

11beta-Hydroxysteroid dehydrogenase in the brain: a novel regulator of glucocorticoid action?

Corticosteroids (glucocorticoids and mineralocorticoids) have multiple actions in the brain which are mediated via specific intracellular receptors. Recently, a novel and important control of glucocorticoid action has been identified in peripheral tissues; prereceptor metabolism by 11beta-hydroxysteroid dehydrogenase (11beta-HSD). This enzyme catalyses the conversion of the active glucocorticoids corticosterone and cortisol to inert 11 keto-products (11-dehydrocorticosterone, cortisone), thus regulating access of glucocorticoids to receptors. Two distinct isozymes occur. 11beta-HSD-1 is a widespread, NADP(H)-dependent enzyme, which shows bidirectional activity in tissue homogenates and microsomal preparations, but may predominantly function as an 11beta-reductase (regenerating active glucocorticoids) in intact cells. 11beta-HSD-2 is a much higher affinity, NAD-dependent, exclusive 11beta-dehydrogenase (glucocorticoid inactivating enzyme), which, when colocalized with otherwise nonselective mineralocorticoid receptors (MR), ensures selective access for aldosterone in vivo. Accumulating evidence indicates widespread expression of 11beta-HSD-1 in the brain. The highest levels are found in cerebellum, hippocampus, cortex, and pituitary, but detectable activity is also present in the hypothalamus (including the paraventricular nucleus) and other regions of neuroendocrine interest. 11beta-HSD-1 protein has been detected on Western blots of brain and immunostaining is widespread, localized predominantly in neurons and their processes. The mRNA encoding 11beta-HSD-1 is also widely expressed in the brain, its distribution broadly paralleling enzyme bioactivity and immunostaining. 11beta-HSD-1 expression is regulated during late prenatal and postnatal ontogeny and by glucocorticoids and stress, prompting suggestions that this isoform may play a role in protecting the brain from the deleterious consequences of glucocorticoid excess. However, in primary cultures of hippocampal neurons, 11beta-HSD-1 functions as a predominant 11beta-reductase, reactivating inert corticoids and thus potentiating neurotoxicity. The functions of 11beta-HSD-1 in the CNS are not defined, but may relate to mood, neuronal survival, and glucocorticoid feedback. The identification of aldosterone-selective actions in the brain (upon blood pressure and salt appetite) predict the presence of 11beta-HSD-2. This isozyme has very limited expression in the adult brain, probably confined to the subregions of the brain stem and the subcommissural organ, where some aldosterone-selective actions may be mediated. However, the midgestation fetal brain highly expresses 11beta-HSD-2, which might modulate glucocorticoid effects on CNS development. Studies with licorice-derived enzyme inhibitors indicate functional effects for 11beta-HSD in the adult brain, notably in the periventricular hypothalamus and limbic system. Thus, 11beta-HSD represents a novel and potentially important level of control of glucocorticoid action in the CNS. Enzyme modulation by pharmacological or other agents may provide a useful means to target increased or attenuated glucocorticoid action to specific sites in the brain.     

Hemispheric control of motor function: a whole brain echo planar fMRI study

A sample of 130 patients with vertical maxillary hyperplasia; mandibular hypoplasia with a high mandibular plane angle; narrow, tapered maxillary dental arch form; and anterior vertical open bite were collected from three different institutions to evaluate the stability of transverse maxillary arch dimensions after correction of the open bite. Surgical treatment consisted of Le Fort I or bimaxillary osteotomies. Intermolar, interpremolar, and anterior arch widths were measured three-dimensionally on dental casts using a Reflex microscope, and transverse stability after orthodontic or surgical maxillary expansion was analyzed. Orthodontic expansion followed by a one-piece Le Fort I intrusion osteotomy was performed in 77 patients, and surgical maxillary expansion by a multisegment Le Fort I intrusion osteotomy was performed in 53 patients. The increase of transverse arch width and the relapse after orthodontic or surgical expansion were not significantly different. The transverse arch width in these two groups did not relapse in 20% of the patients after a mean follow-up of 69 months. An additional bilateral sagittal split osteotomy had no detectable effect on stability. Patients who underwent a multisegment Le Fort I osteotomy stabilized with rigid internal fixation showed better transverse stability than those with intraosseous wire fixation and maxillomandibular fixation. Maxillary intermolar and interpremolar arch width relapses were not correlated with tongue interposition or loss of interdigitation. The relapse of these arch widths showed significant correlations with clockwise rotation of the mandible but not with changes of overbite or overjet.     

The central adaptation syndrome: psychosocial stress as a trigger for adaptive modifications of brain structure and brain function

This review makes an attempt to combine data from biological and psychosocial stress literature and to suggest an alternative interpretation of the relationship between stress and disease. It rearranges the presently available knowledge on the short- and long-term effects of stress on many different aspects of brain structure and brain function in the form of a new conceptualization of the biological role of the stress response. The higher associative brain structures are not only the sites in which environmental and psychosocial demands are recognized and from which a less or more systemic, i.e. controllable or uncontrollable, stress response is initiated. They are also the sites which are primarily affected in the course of the stress response: the stress response acts as a trigger for the adaptive modification of the structure and the function of the brain of higher vertebrates and serves thus to adjust, in a self-optimizing manner, the behavior of an individual to the ever-changing requirements of its external world. This novel concept summarizes a large amount of information into a framework that lends itself to testable strategies for future research.     

Glutamate receptors: brain function and signal transduction

Glutamate receptors are important in neural plasticity, neural development and neurodegeneration. N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors act as glutamate-gated cation channels, whereas metabotropic receptors (mGluRs) modulate the production of second messengers via G proteins. Molecular studies from our and other laboratories indicated that NMDA receptors and mGluRs exist as multiple subunits (NMDAR1 and NMDAR2A-2D) and multiple subtypes (mGluR1-mGluR8). In light of the molecular diversity of glutamate receptors, we explored the function and intracellular signaling mechanisms of different members of glutamate receptors. In the visual system, retinal bipolar cells receive glutamate transmission from photoreceptors and contribute to segregating visual signals into ON and OFF pathways. The molecularly cloned mGluR6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. Gene targeting of mGluR6 results in a loss of ON responses without changing OFF responses and severely impairs detecting visual contrasts. Since AMPA receptors mediate OFF responses in OFF-bipolar cells, two distinct types of glutamate receptors effectively operate for ON and OFF responses. mGluR1 and mGluR5 are both coupled to inositol triphosphate (IP3)/calcium signal transduction with an identical agonist selectivity. Single-cell intracellular calcium ([Ca2+]i) recordings indicated that glutamate evokes a non-oscillatory and oscillatory [Ca2+]i response in mGluR1-expressing and mGluR5-expressing cells, respectively. This difference results from a single amino acid substitution, aspartate of mGluR1 or threonine of mGluR5, at the G protein-interacting carboxy-terminal domains. Protein kinase C phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2+]i oscillations in mGluR5-expressing cells and cultured glial cells. Thus, the two closely related mGluR subtypes mediate diverging intracellular signaling in glutamate transmission.     

Aging of brain and the maintenance of the function

The chicken karyotype comprises 39 chromosome pairs of which at least 29 are 'microchromosomes'. Microchromosomes account for about 25% of the genomic DNA, but they are cytologically indistinguishable from one another (1). Due to technical limitations there is a strong bias of mapped genes within the chicken genome database ChickGBASE (2) towards macrochromosomes 1-6 and Z, with specific assignments to only one microchromosome (3,4). Several genes have, however, been assigned to the microchromosome group as a whole (3,5-9), demonstrating that these tiny chromosomes do not represent genetically inert DNA. To determine the overall chromosomal distribution of genes, as well as to provide a mapping resource, we prepared a CpG island library from chicken using differential binding to a methyl-CpG chicken using differential binding to a methyl-CpG binding column before and after de novo methylation (10). Surprisingly, we found that chicken CpG islands are highly concentrated on the microchromosomes, whereas macrochromosomes 1-6 are comparatively gene-poor by this assay. Our results raise the possibility that gene density on chicken microchromosomes approaches the maximum value known for vertebrates.     

Structure and function of a novel ErbB ligand, NTAK

A novel member of the epidermal growth factor (EGF) family, the neural and thymus-derived activator for ErbB kinase (NTAK) has been cloned from the cDNA library of a rat pheochromocytoma cell line, PC12 cells and human neuroblastoma cell line, SK-N-SH cells. Four alternative spliced isoforms from rat cDNA have been detected by the methods of RT-PCR. The rat NTAK alpha 2a isoform exhibits 94% identity in its sequence with the human NTAK alpha isoform. Three characteristic Ig-like, EGF-like and hydrophobic domains have been identified in rat and human NTAK molecules. Recombinant NTAK, the soluble 46 kDa form, binds directly to ErbB3 and ErbB4, but not ErbB1 and B2. NTAK, however, transactivates with heterodimer such as ErbB1/B3, B1/B4, B2/B3, B2/B4, and B3/B4. NTAK stimulates the differentiation of MDA-MB-453 cells, derived from blast carcinoma. NTAK competitively inhibits the binding of [125I] NRG-1 to these cells. Thus, NTAK is a new member of the EGF family displaying NRG-1 properties.     

Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores

In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe2+-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidylcholine (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Platelet-activating factor: a case for its role in CNS function and brain injury

Specially designed Thor-Lo footwear has been shown to reduce the in-shoe foot pressures in diabetic patients at risk of foot ulceration when compared to their own footwear. Fifty at high risk patients 32 (64%) males, 17 (34%) type 1 diabetes) have been provided with this foot wear and have been followed up for 6 months. Mean age was 57.6 (range, 34-78) years, duration of diabetes 22.4 (range, 4-50) years, Neuropathy Symptom Score 3.36 +/- 2.96 (mean +/- S.D.), Neuropathy Disability Score 16.8 +/- 6.83, VPT 43.4 +/- 11.8 Volts while 43 (86%) could not feel a 5.07 or smaller Semmes-Weinstein monofilament. Forty-two (84%) patients were re-examined at an interim visit 3 months after baseline, while 37 (74%) completed the study. In-shoe peak forces and pressures were measured using the F-Scan system. No difference was found among the peak force among baseline (95.5 +/- 26 kg), interim (96.5 +/- 33) and final visit (97.7 +/- 25.2, P + NS). There was no difference in peak pressures at the baseline (3.98 +/- 1.42 kg.cm-2), second visit (4.13 +/- 2.30) and the final visit (4.25 +/- 1.51). Nine (18%) patients developed foot problems and one died during the study. We conclude that no changes in foot pressures were found over a period of 6 months of continuous usage of the specially designed footwear in a group of diabetic patients at risk of foot ulceration. Further prospective studies are required to evaluate the impact of specially designed footwear in reducing the rate of foot ulceration.     

Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation. structure-function relationships in polytopic membrane proteins.     

3H]Alnespirone: a novel specific radioligand of 5-HT1A receptors in the rat brain

Determination of the optimal assay conditions for the specific binding of a tritiated derivative of the novel potential anxiolytic drug alnespirone (S-20499, (+)-4-[N-(5-methoxy-chroman-3-yl)-N-propylamino]butyl-8-azaspiro-( 4,5)-decane-7,9-dione) allowed the demonstration that this radioligand bound with a high affinity (Kd = 0.36 nM) to a homogeneous class of sites in rat hippocampal membranes. The pharmacological properties of [3H]alnespirone specific binding sites matched exactly (r = 0.95) those of 5-HT1A receptors identified with [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) as radioligand. Furthermore, membrane binding experiments and autoradiographic labeling of tissue sections showed that the regional distribution of [3H]alnespirone specific binding sites in the rat brain and spinal cord superimposed over that of 5-HT1A receptors specifically labeled by [3H]8-OH-DPAT. However, the differential sensitivity of [3H]alnespirone and [3H]8-OH-DPAT specific binding to various physicochemical effectors (temperature, pH, Mn2+, N-ethyl-maleimide) supports the idea that these two agonist radioligands did not recognize 5-HT1A receptors exactly in the same way. These differences probably account for the reported inability of alnespirone, in contrast to 8-OH-DPAT, to induce some 5-HT1A receptor-mediated behavioural effects in rats.