Membrane proteins: the 'Wild West' of structural biology

Historically, the task of determining the structure of membrane proteins has been hindered by experimental difficulties associated with their lipid-embedded domains. Here, we provide an overview of recently developed experimental and predictive tools that are changing our view of this largely unexplored territory - the 'Wild West' of structural biology. Crystallography, single-particle methods and atomic force microscopy are being used to study huge membrane proteins with increasing detail. Solid-state nuclear magnetic resonance strategies provide orientational constraints for structure determination of transmembrane (TM) alpha-helices and accurate measurements of intramolecular distances, even in very complex systems. Longer distance constraints are determined by site-directed spin-labelling electron paramagnetic resonance, but current labelling strategies still constitute some limitation. Other methods, such as site-specific infrared dichroism, enable orientational analysis of TM alpha-helices in aligned bilayers and, combined with novel computational and predictive tools that use evolutionary conservation data, are being used to analyze TM alpha-helical bundles.     

STRUCTURE AND FUNCTION OF BENTHIC ALGAL COMMUNITIES IN AN EXTREMELY ACID RIVER1

The composition of algal species and pigments and the structural and functional characteristics of the algal community were investigated in an acid stream of southwestern Spain, the Río Tinto. The algal community had low diversity and showed few seasonal differences. It was mainly made up of Klebsormidium flaccidum Kütz. (Silva, Mattox & Blackwell) that produced long greenish or purplish filaments, Pinnularia acoricola Hust. (producing brown patches) and Euglena mutabilis Schmitz. The algal filaments made up a consistent biofilm that also included fungal hyphae, iron bacterial sheaths, diatoms, and mineral particles. HPLC analyses on Río Tinto samples showed that undegraded chl accounted for 67% of the total chl in the filamentous patches but were a minority in the brown patch (2.6%). The brown patch had a concentration of carotenoids eight times lower than that observed in the green patch. When chl concentrations were weighted for the proportion of the different patches on the streambed, undegraded chl a accounted for 89.2 mg chl a·m ^{? 2} of stream surface area (5.4 g C·m ^{? 2}). This high algal biomass was supported by relatively high nutrient concentrations and by a high phosphatase activity (V_max = 137.7 nmol methylumbelliferyl substrate·cm ^{? 2}·h ^{? 1} 1 Received 15 July 2002. Accepted 17 February 2003. , K_m = 0.0045 μM). The remarkable algal biomass in Río Tinto potentially contributed to the bacterial–fungal community and to the macroinvertebrate community and emphasizes the role that the algae may have in the organic matter cycling and energy flow in extreme systems dominated by heterotrophic microorganisms.     

Identification of the autoantigen HB as the barrier-to-autointegration factor

The HB autoantigen, a 10-kDa DNA-binding protein recognized by autoantibodies only when bound to DNA, was identified by two-dimensional electrophoresis. Silver-stained protein spots corresponding to the antigen were excised from two-dimensional electrophoresis gels, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization-reflectron time of flight and nano-electrospray ionization-ion trap/mass spectrometry. Data base search identified the HB antigen as the barrier-to-autointegration factor, a cellular protein implicated in the cellular cycle that blocks autointegration and promotes intermolecular integration of retrovirus such as the Moloney murine leukemia and the human immunodeficiency type 1 virus. The physicochemical characteristics described for these proteins, their ability to bind double-stranded DNA but not single-stranded DNA, and their nuclear localization confirm that HB and barrier-to-autointegration factor are the same protein.     

Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.     

Effects of oral aluminum on essential trace elements metabolism during pregnancy

The present study was conducted to assess in rats the effects of oral aluminum (Al) exposure on calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), and iron (Fe) accumulation and urinary excretion. Three groups of plug-positive Sprague-Dawley (SD) rats were given by gavage 0, 200, and 400 mg/kg/d of Al(OH)₃ on gestational days 1–20. Three groups of nonpregnant female SD rats of the same age received Al(OH)₃ by gavage at the same doses for 20 consecutive days. At the end of the treatment period, 24-h urine samples were collected for analysis of Al and essential elements. Subsequently, all animals were sacrificed and samples of liver, bone, spleen, kidneys, and brain were removed for metal analyses. With some exceptions, the urinary amounts of Al, Mn, and Cu excreted by pregnant animals as well as the urinary levels of Al excreted by nonpregnant rats were higher in the Al-treated groups than in the respective control groups. Although higher Al levels were found in the liver of pregnant rats, the concentrations of Al in the brain of these animals were lower than those found in the same tissues of nonpregnant rats. With regard to the essential elements, tissue accumulation was most affected in pregnant than in nonpregnant animals. In pregnant rats, the hepatic and renal concentrations of Ca, Mg, Mn, Cu, Zn, and Fe, as well as the levels of Ca in bone, and the concentrations of Cu in brain were significantly higher in the Al-exposed groups than in the control group. According to the current results, oral Al exposure during pregnancy can produce significant changes in the tissue distribution of a number of essential elements.     

Cardiovascular effects of peptide kinin B2 receptor antagonists in rats

Bradykinin (BK) is a vasoactive peptide reputed to play an important role in cardiovascular homeostasis. In this study, we describe the cardiovascular changes (mean blood pressure (BP) and heart rate (HR)) induced by the i.v. administration (left jugular vein) of two selective kinin B2 receptor antagonist, namely icatibant (0.1-1 micromol/kg as a bolus) and MEN1 1270 (0.1-1 micromol/kg as a bolus or 1 micromol/kg infused in 15 or 60 min), in urethane-anaesthetized or conscious rats with an indwelling catheter implanted in the right carotid artery for BP measurements. In conscious rats, icatibant at 0.1 or 0.3 micromol/kg did not change BP but at 0.1 micromol/kg increased HR at 30 min from administration. MEN1 1270 at 0.1 or 0.3 micromol/kg induced a dose-related increase in BP and a concomitant bradycardia (significant at 0.3 micromol/kg) lasting for 5 or 30 min, respectively. Icatibant at 1 micromol/kg induced a slight (P < 0.05) increase in BP that resolved in 5 min and a biphasic tachycardia (peaks at 30 and 90 min from administration). MEN1 1270 at 1 micromol/kg induced a triphasic change in HR (tachycardia in the first 5 min, bradycardia at 30 min, and tachycardia at 90 and 120 min) and a biphasic change in BP (hypotension at 15 min and hypertension at 30 min). The i.v. infusion of MEN1 1270 (1 micromol/kg in 15 or 60 min) produced hypertension, whereas HR was increased only following the 15-min infusion. In urethane-anaesthetized rats, both icatibant and MEN1 1270 (0.1 micromol/kg as a bolus) increased BP and the onset for this effect was correlated with the time course of the antagonism of BK-induced hypotension, where the effect of MEN1 1270 was more rapid than that of icatibant. These results indicate that kinin B2 receptor antagonists can induce acute cardiovascular effects, and the reason for the different haemodynamic profile between icatibant and MEN1 1270 could be putatively attributed to kinetic characteristics.     

Paraoxonase1-192 polymorphism modulates the effects of regular and acute exercise on paraoxonase1 activity

Regular exercise practise is a protective factor against coronary heart disease and enhances antioxidant systems, whereas acute exercise appears to be a major source of increased oxidative stress. Paraoxonase1 (PON1) is an antioxidant HDL-linked enzyme, whose activity toward paraoxon (PON1 activity) is strongly modulated by the PON1-192 polymorphism, comprising Q and R alleles for low and high PON1 activity, respectively. Another polymorphism at the PON1 locus, the PON1-55, modulates PON1 protein and activity levels. PON1 activity, lipid levels, and oxidized LDL concentration were determined in 17 healthy young volunteers before and after a 16-weeks aerobic exercise training period. Furthermore, PON1 activity was analyzed after a bout of exercise in both situations. We found that regular exercise was associated with a decrease in oxidized LDL levels, and an increase in PON1 activity in QQ subjects and with a decrease in PON1 activity in R carriers. A bout of exercise produced an increase in PON1 activity just after the bout of exercise, followed by a decrease in its activity. A recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. These results suggest that the effects of regular and acute exercise on PON1 activity levels are modulated by PON1-192 polymorphism. Changes were less evident for the PON1-55 polymorphism.     

The peptide repertoires of HLA-B27 subtypes differentially associated to spondyloarthropathy (B*2704 and B*2706) differ by specific changes at three anchor positions

HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg(2) and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and POmega-2 and impaired binding of Tyr and Arg at POmega. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and POmega-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.     

Ontogenesis of the vitamin D receptor in rat heart

1,25-Dihydroxyvitamin D(3) through its receptor (vitamin D receptor; VDR) has important physiological effects such as calcium transport and cell growth and differentiation. Although the VDR is present in a variety of cell lines as well as in numerous tissues, including rat and human heart, no data are available about the presence of VDR in heart at different steps of rat life. In this study we evaluated the VDR expression using RT-PCR and immunohistochemical techniques in fetal (17, 18 and 20 gestational days), neonatal (4 and 8 days) and adult rat heart. Immunohistochemical techniques showed the VDR protein localisation in the nuclei of cardiac muscle fibres. Also, we demonstrated that VDR mRNA expression is changing over these different periods of development, showing significant differences in 20 days versus 18 days of fetal age. These changes in VDR expression may be related to other parameters associated with the development of the cardiac muscle and/or intracellular cardiac cell calcium homeostasis.