Inflammatory bowel disease and the genes for the natural resistance-associated macrophage protein-1 and the interferon-Á receptor 1

The genes for the interferon-γ receptor1 and the natural resistance-associated macrophage protein1 (NRAMP1) control the immune response to intracellular microbial pathogens. Such pathogens, in particular Mycobacterium paratuberculosis, have been implicated in the pathogenesis of Crohn's disease. We studied markers in the genes for NRAMP1 and two mutations in the interferon-γ receptor in relation to inflammatory bowel disease (IBD) in the following groups: 270 healthy individuals, 74 patients with Crohn's disease, 72 patients with ulcerative colitis, and 40 patients with primary sclerosing cholangitis. We studied the allele frequencies of two restriction fragment length polymorphisms in the gene for NRAMP1 and the prevalence of two mutations in the interferon-γ receptor1 gene. The markers in the NRAMP1 gene were not associated with inflammatory bowel disease. Also, the mutations in the interferon-γ receptor1 were not found in the 186 IBD patients. Genetic markers in NRAMP1 are thus not associated with IBD. Therefore this gene is not likely to play a role in the pathogenesis of IBD. The mutation in the interferon-γ receptor was not found in our IBD patients group. Accepted: 13 October 1998     

Does the plasma endothelin-1 concentration reflect atherosclerosis in the elderly?

BACKGROUND AND OBJECTIVE: Recently, attention has been focused on endothelin-1 (ET-1) as an indicator of atherosclerosis. However, normal levels of ET-1 are frequently found in elderly patients. We investigated the relationships between echocardiographic findings and ET-1 in 117 inpatients for rehabilitation. METHODS: The patients were 34 men and 83 women, 83.4 +/- 0.8 years old (mean +/- SE), with the following diseases: cerebrovascular diseases (n = 83), cardiovascular diseases of New York Heart Association Class I or II (n = 57), diabetes mellitus (n = 11) and hyperlipidemia (n = 12). Ejection fraction (EF) and left ventricular mass index (LVMI) were calculated by echocardiographic studies. RESULTS: The average ET-1 was within the normal range, but a significant positive correlation was observed between age and ET-1 (ET-1 = 1.8 +/- 0.1 pg/ml, r = 0. 248, p < 0.01). Moreover, plasma ET-1 was positively correlated to total cholesterol and to low density lipoprotein cholesterol in patients with a history of hypertension (n = 41, r = 0.318 and r = 0. 314, both p < 0.05). Gender was not a significant factor. There was no significant difference in ET-1 between patients with mild renal dysfunction (n = 54) and patients with normal renal function (n = 63) (blood urea nitrogen = 30.9 +/- 2.4 vs. 15.1 +/- 0.5 mg/dl, serum creatinine = 1.03 +/- 0.06 vs. 0.59 +/-0.02 mg/dl, ET-1 = 1.7 +/- 0.1 vs. 1.8 +/- 0.1 pg/ml), or between patients with (n = 50) and without (n = 67) left ventricular hypertrophy (LVMI = 146 +/- 6 vs. 83 +/- 2 g/m(2), ET-1 = 1.8 +/- 0.1 vs. 1.8 +/- 0.1 pg/ml). There was no difference in ET-1 between patients with EF <50% (n = 27) and EF >/=50% (n = 90) (ET-1 = 1.8 +/- 0.2 vs. 1.7 +/- 0.1 pg/ml). CONCLUSION: There were no significant relationships between echocardiographic findings and ET-1 in elderly subjects without overt heart failure. However, in patients with a history of hypertension, measuring ET-1 may be useful for estimating the extent of atherosclerosis.     

Netrin-1–α3β1 integrin interactions regulate the migration of interneurons through the cortical marginal zone

Cortical GABAergic interneurons, most of which originate in the ganglionic eminences, take distinct tangential migratory trajectories into the developing cerebral cortex. However, the ligand–receptor systems that modulate the tangential migration of distinct groups of interneurons into the emerging cerebral wall remain unclear. Here, we show that netrin-1, a diffusible guidance cue expressed along the migratory routes traversed by GABAergic interneurons, interacts with α3β1 integrin to promote interneuronal migration. In vivo analysis of interneuron-specific α3β1 integrin, netrin-1–deficient mice (α3^lox/−Dlx5/6-CIE, netrin-1^−/−) reveals specific deficits in the patterns of interneuronal migration along the top of the developing cortical plate, resulting in aberrant interneuronal positioning throughout the cerebral cortex and hippocampus of conditional α3^lox/−Dlx5/6-CIE, netrin-1^−/− mice. These results indicate that specific guidance mechanisms, such as netrin-1–α3β1 integrin interactions, modulate distinct routes of interneuronal migration and the consequent positioning of groups of cortical interneurons in the developing cerebral cortex.The development of the six-layered cerebral cortex, composed predominantly of glutamatergic projection neurons and GABAergic interneurons, depends on the appropriate migration of neurons from ventricular zones of the dorsal and ventral telencephalon to their final position in the emerging cortical plate. Neuronal progenitors from the dorsal telencephalon migrate radially as cohorts along the radial glial scaffold, pass their predecessors in the developing cortical plate, and coalesce into layers, giving rise to glutamatergic projection neurons (1). In contrast, neuronal precursors from the ventral telencephalon, principally the medial ganglionic eminence (MGE), migrate tangentially through the cortical marginal zone and intermediate zone and enter the cortical plate, using the radial glial scaffold, before differentiating into GABAergic interneurons (2–4). A subset of these interneurons initially migrates radially inwards, toward the cortical ventricular zone, before turning back up toward the cortical plate (4). These different modes of neuronal migration are coordinated temporally to achieve the “inside-out” laminar organization of neurons in the neocortex.Specific cell adhesion interactions between neurons, glia, and the surrounding ECM are critical for the appropriate migration and placement of cortical neurons. These adhesive interactions are mediated in large part by integrins, heterodimeric cell-surface ECM receptors, which serve as structural links between extracellular ligands and the internal cytoskeleton. The functional significance of integrin signaling for cerebral cortical development is demonstrated by the distinct types of cortical malformations exhibited by mice deficient in α_v, α₃, α₆, β₁, and β₄ integrins (1, 5–8). However, the selective adhesive interactions needed to coordinate the migration of distinct groups of interneurons and projection neurons into the developing cerebral cortex remain poorly understood.The prominent expression of netrin-1 in the ganglionic eminence (GE), where interneurons originate (9), as well as the expression of α3β1 integrin in migrating cortical neurons (5), suggests that netrin-1–α3β1 integrin interactions may influence specific aspects of the tangential migration of GABAergic interneurons. Recent evidence that α3β1 integrin serves as a receptor for netrin-1 in epithelial cells and evidence for cross-talk between integrins and classical netrin receptors have opened an avenue for investigating this possibility (10). Netrins serve as secreted guidance cues, regulating axonal outgrowth and neuronal migration by binding to Deleted in Colon Cancer (DCC), Down''s Syndrome Cell Adhesion Molecule (DSCAM), or UNC5 receptors (11, 12). Netrin-1 interacts with DCC and DSCAM to induce growth cone attraction (12, 13), whereas its association with UNC5 receptors promotes axonal repulsion (14). Recently, Yebra and colleagues (10) have demonstrated that α6β4 and α3β1 integrins regulate the migration of epithelial cells on netrin-1 through a direct interaction of these integrins with the C-terminal domain of netrin-1, although the significance of these interactions is yet to be determined in vivo. Here, we have investigated the contribution of netrin-1–α3β1 integrin–mediated signal transduction to the migration of GABAergic interneurons from the MGE into the developing cerebral wall.     

Caging the beast: TRIM5α binding to the HIV-1 core

The potent HIV-1 inhibitor TRIM5α blocks HIV-1 infection by accelerating the uncoating of HIV-1. TRIM5α is known to form higher-order self-association complexes that contribute to the avidity of TRIM5α for the HIV-1 capsid, and are essential to inhibit infection; these higher-order self-association complexes are dependent upon an intact B-box 2 domain. Even though the ability to form higher-order self-association complexes resembles the clathrin triskelion that forms a protein array, or cage, around the endocytic vesicle, evidence for the ability of TRIM5α to assemble a similar type of structure surrounding the HIV-1 core has been lacking. Recent work by Ganser-Pornillos, Chandrasekaran and colleagues has now demonstrated the ability of the restriction factor TRIM5α to "cage" or "net" the HIV-1 core by forming an hexagonal array on the surface of the viral capsid. This hexagonal array is strikingly similar in design to the array formed by the clathrin triskelion on the surface of the clathrin-coated endocytic vesicle. This remarkable finding represents an important advance on our understanding of the restriction factor TRIM5α, and suggests that TRIM5α cages the HIV-1 core in order to terminate infection. The present note discusses the implications of this discovery.     

Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor

The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1–4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors.     

G-protein-mediated inhibition of the Trp channel TRPM1 requires the Gβγ dimer

ON bipolar cells are critical for the function of the ON pathway in the visual system. They express a metabotropic glutamate receptor (mGluR6) that, when activated, couples to the G(o) class of G protein. The channel that is primarily responsible for the synaptic response has been recently identified as the transient receptor potential cation channel subfamily M member 1 (TRPM1); TRPM1 is negatively coupled to the mGluR6/Go cascade such that activation of the cascade results in closure of the channel. Light indirectly opens TRPM1 by reducing transmitter release from presynaptic photoreceptors, resulting in a decrease in mGluR6 activation. Conversely, in the dark, binding of synaptic glutamate to mGluR6 inhibits TRPM1 current. Closure of TRPM1 by G-protein activation in the dark is a critical step in the process of ON bipolar cell signal transduction, but the precise pathway linking these two events is not understood. To address this question, we measured TRPM1 activity in retinal bipolar cells, in human ependymal melanocytes (HEMs) that endogenously express TRPM1, and in HEK293 cells transfected with TRPM1. Dialysis of the Gβγ subunit dimer, but not Gα(o), closed TRPM1 channels in every cell type that we tested. In addition, activation of an endogenous G-protein-coupled receptor pathway in HEK293 cells that releases Gβγ without activating Go protein also closed TRPM1 channels. These results suggest a model in which the Gβγ dimer that is released as a result of the dissociation from Gα(o) upon activation of mGluR6 closes the TRPM1 channel, perhaps via a direct interaction.     

Are the antagonists of angiotensin II AT1 receptors protectors of the kidney?]

A lot of evidence points to the important role of the renin-angiotensin system in the physiopathology of hypertension and the progression of chronic renal failure. In this review, the authors report the data concerning the protective effects of antagonists of angiotensin II AT1 receptors (AT1ra). The AT1 ra have been shown to have beneficial effects in most experimental models of nephropathy in which they have been tested (renal ischaemia, essential or induced hypertension, glomerulonephritis, 5/6 nephrectomy, renal transplantation, induced diabetes, toxic and radiotherapy-induced nephropathy). Clinical trials confirm these beneficial effects. In healthy subjects and hypertensive patients, the AT1 ra have identical effects to those of angiotensin converting enzyme (ACE) inhibitors on renal haemodynamics. In hypertensives, Candesartan and Irbesartan increase renal blood flow and the glomerular filtration rate and decrease the filtration fraction. Two studies have also shown that Candesartan and Irbesartan reduce proteinuria in diabetic patients. Similar results have been reported in essential hypertension with renal failure. These data suggest that AT1 ra have beneficial effects on the progression of experimental kidney disease and on proteinuria in the clinical setting. Of the pharmacological agents available for use in this class, it is essential to propose molecules whose efficacy in antagonising the effects of angiotensin II lasts throughout the 24 hour period. Clinical trials are under way to evaluate the effects of AT1 ra on renal function in man over a long period.     

Backtracking on the folding landscape of the β-trefoil protein interleukin-1β?

Interleukin-1β (IL-1β) is a cytokine within the β-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the β-bulge, residues 47–53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1β, a β-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.     

A haplotype derived from the common variants at the −1997G/T and Sp1 binding site of the COL1A1 gene influences risk of postmenopausal osteoporosis in India

The aim of the present study was to investigate the association between Collagen 1 alpha 1 (COL1A1) polymorphism and osteoporosis in DEXA verified 349 (145 osteoporotic, 87 osteopenic and 117 normal) postmenopausal women of India, who were not taking hormone replacement therapy. Two single-nucleotide polymorphisms (SNPs), that is, ?1997G/T (rs1107946) and +1245G/T (rs1800012, Sp1) of the COL1A1 gene, were analyzed. Minor allele frequencies of rs1107946 and rs1800012 were 0.15 and 0.20 in osteoporotic women, 0.18 and 0.18 in osteopenic and 0.20 and 0.17 in women having normal bone mass. An allele dose effect with BMD of lumbar spine has been exhibited by major allele G of rs1107946 (GG: 0.86 g/cm², GT: 0.91 g/cm² and TT: 0.93 g/cm²) and minor allele T of rs1800012 (GG: 0.91 g/cm², GT: 0.87 g/cm² and TT: 0.81 g/cm²). Disease association analysis revealed a haplotype GT that confers approximately threefold higher risk of osteoporosis in the carriers (OR 3.12, 95% CI 1.24–8.88, P = 0.008) after adjusting the confounding effect of age, BMI and years since menopause. These results suggest that GT haplotype of COL1A1 gene is associated with a higher risk of postmenopausal osteoporosis in Northwest Indian women.     

Antibiotic treatment partially protects against type 1 diabetes in the Bio-Breeding diabetes-prone rat. Is the gut flora involved in the development of type 1 diabetes?

Aims/hypothesis Accumulating data suggest that the gut immune system plays a role in the development of type 1 diabetes. The intestinal flora is essential for the development of the (gut) immune system and the establishment of tolerance. It has been reported that oral administration of food and bacterial antigens early in life suppresses later development of diabetes in the Bio-Breeding diabetes-prone (BB-DP) rat. This study was designed to investigate the possible relationship between the development of diabetes and the composition of intestinal flora.Materials and methods The intestinal flora of BB-DP rats, a rat model for type 1 diabetes, was characterised long before the clinical onset of diabetes by fluorescent in situ hybridisation. In a separate experiment, BB-DP rats were treated with antibiotics and the effect on diabetes incidence and level of insulitis was analysed.Results We observed a difference in bacterial composition between rats that eventually did and those that did not develop diabetes. This difference was detectable long before clinical onset of the disease. Rats that did not develop diabetes at a later age displayed a lower amount of Bacteroides sp. Modulation of the intestinal flora through antibiotic treatment decreased the incidence and delayed the onset of diabetes. A combination of antibiotic treatment and a protective hydrolysed casein diet completely prevented diabetes in the BB-DP rat.Conclusions/interpretation Our data suggest that the intestinal flora is involved in the development of type 1 diabetes. Factors influencing composition of the intestinal flora could be a target for therapeutic intervention.     

Folding mechanism of the metastable serpin α1-antitrypsin

The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded β-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α(1)-antitrypsin (α(1)-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, β-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into β-sheets B and C is largely complete before the center of β-sheet A begins to fold. We propose this as the mechanism for trapping α(1)-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α(1)-AT avoids polymerization during folding.