Does inbreeding affect N-glycosylation of human plasma proteins?

Inbreeding depression and heterosis are the two ends of phenotypic changes defined by the genome-wide homozygosity. The aim of this study was to investigate the association of genetic marker-based homozygosity estimates with 46 N-glycan features measured in human plasma. The study was based on a total of 2,341 subjects, originating from three isolated island communities in Croatia (Vis and Korcula islands) and Scotland (Orkney Islands). Inbreeding estimates were associated with an increase in tetrantennary and tetrasialylated glycans, and a decrease in digalactosylated glycans (P?相似文献   

Do plasma proteins distinguish between liposomes of varying charge density?

Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a “protein corona” onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density.     

Genetic studies of low-abundance human plasma proteins. I. Microheterogeneity of zinc-α2-glycoprotein in biological fluids

A high-resolution isoelectric focusing technique followed by immunoblotting has been utilized to determine the microheterogeneity of zinc-alpha 2-glycoprotein in a large number of plasma samples from U.S. Caucasians, Blacks, and Eskimos. With the exception of one Black individual, all samples were found to contain an invariant multiple-banded pattern which, after desialylation, was reduced to a single band, suggesting that the microheterogeneity observed is due to differences in the sialic acid content of a single protein product. The asialo forms of the variant sample consist of two distinct bands, consistent with the occurrence of a rare genetic variant at the zinc-alpha 2-glycoprotein structural locus. Unfortunately family studies were not feasible. In addition to plasma, the present technique has been applied to detection of zinc-alpha 2-glycoprotein microheterogeneity in amniotic fluid, saliva, and tears. The amniotic fluid pattern is identical to that present in plasma. However, the patterns observed in saliva and tears are different from each other as well as from that in plasma and could be controlled by separate loci.     

Acute phase proteins are major clients for the chaperone action of α2-macroglobulin in human plasma

Extracellular protein misfolding is implicated in many age-related diseases including Alzheimer's disease, macular degeneration and arthritis. In this study, putative endogenous clients for the chaperone activity of α₂-macroglobulin (α₂M) were identified after human plasma was subjected to physiologically relevant sheer stress at 37 °C for 10 days. Western blot analysis showed that four major acute phase proteins: ceruloplasmin, fibrinogen, α₁-acid glycoprotein and complement component 3, preferentially co-purified with α₂M after plasma was stressed. Furthermore, the formation of complexes between α₂M and these putative chaperone clients, detected by sandwich ELISA, was shown to be enhanced in response to stress. These results support the hypothesis that α₂M plays an important role in extracellular proteostasis by sequestering misfolded proteins and targeting them for disposal, particularly during acute phase reactions.     

Therapeutic effects of systemic administration of chaperone αB-crystallin associated with binding proinflammatory plasma proteins

The therapeutic benefit of the small heat shock protein αB-crystallin (HspB5) in animal models of multiple sclerosis and ischemia is proposed to arise from its increased capacity to bind proinflammatory proteins at the elevated temperatures within inflammatory foci. By mass spectral analysis, a common set of ～70 ligands was precipitated by HspB5 from plasma from patients with multiple sclerosis, rheumatoid arthritis, and amyloidosis and mice with experimental allergic encephalomyelitis. These proteins were distinguished from other precipitated molecules because they were enriched in the precipitate as compared with their plasma concentrations, and they exhibited temperature-dependent binding. More than half of these ligands were acute phase proteins or members of the complement or coagulation cascades. Consistent with this proposal, plasma levels of HspB5 were increased in patients with multiple sclerosis as compared with normal individuals. The combination of the thermal sensitivity of the HspB5 combined with the high local concentration of these ligands at the site of inflammation is proposed to explain the paradox of how a protein believed to exhibit nonspecific binding can bind with some relative apparent selectivity to proinflammatory proteins and thereby modulate inflammation.     

Diverse β subunits of heterotrimeric G proteins are present in thyroid plasma membranes

The functioning of heterotrimeric G protein α subunits in the transduction of hormonal signals to appropriate intracellular responses is well recognized. Much less is known about the distribution of isoforms and functions of G protein β subunits. Here, using specific antibodies, we documented that in plasma membranes of the thyroid cell line Nthy-ori 3-1 all Gβ isoforms-Gβ₁, Gβ₂, Gβ₃, Gβ₄ and Gβ₅ are present, while the Gβ₃ occurs in minute amount. In plasma membrane fraction isolated from pooled postoperative thyroids of patients with nodular goiter and Graves’ disease, the Gβ₁, Gβ₂, Gβ₄ and Gβ₅ subunits were found, whereas Gβ₃ could not be detected.Competition studies revealed that the Gβ₂ is the principal Gβ subunit in membranes from cultured thyroid cells, originated from normal thyroid, as well as in membranes from patients’ thyroids. This suggests that Gβ₂ subunit cooperates with Gα_s subunit, the most active of the Gα variants, during stimulation of adenylate cyclase which constitutes the main route of physiological thyroid stimulation.     

Effect of prostaglandins E2 and F2α on in vitro development and hatching of caprine blastocysts

Prostaglandin E₂ has been shown to increase the ovine embryo hatching rate, and PGF_2α to reduce the development of rabbit, bovine, and rat embryos. The objective was to determine the effects of PGE₂ and PGF_2α on development of caprine embryos. Estrus was synchronized in does (n = 25) with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days, and superovulated with 20 units of FSH. On day 6 following estrus, embryos were flushed (n = 128) and incubated individually per well in 25 μl droplets of TCM-199 and BSA (8 mg/ml) for 6 days at 38.5 °C in a 5% CO₂: air with one of the following treatments: (1) control (0.0002% EtOH), (2) PGE₂ (7 ng/ml), (3) PGF_2α (7 ng/ml), (4) low PGE₂:high PGF_2α (3.5 ng/ml:14 ng/ml), (5) balanced PGE₂:PGF_2α (7 ng/ml:7 ng/ml), or (6) high PGE₂:low PGF_2α (14 ng/ml:3.5 ng/ml). Treatment with PGE₂ alone reduced (P < 0.05) the hatching rate (1/15; 7%). The hatching rate of embryos treated with PGF_2α alone (9/18; 50%), low PGE₂:high PGF_2α (8/16; 50%), and balanced PGE₂:PGF_2α (11/16; 69%) were similar to control (6/18; 33%). In contrast, the hatching rate was non-significantly increased (13/18; 72%) with the high PGE₂:low PGF_2α treatment. None of the treatments affected development from the morula to blastocyst stage. From the current data, it can be concluded that PGE₂ alone reduced hatching rate, and PGF_2α alone had no effect on the development of caprine embryos. High concentrations of PGE₂ with PGF_2α improved the hatching rates. Thus, uterine concentrations of PGE₂ may need to reach a threshold level to improve embryo hatching, as previously reported, while increased uterine concentrations of PGF_2α during early pregnancy would not affect development of the embryo.     

Effect of prostaglandins F2α and E2 on sensitivity of rabbit cortical neurons to acetylcholine and noradrenalin

The effect of prostaglandins F₂ and E₂ on unit activity and sensitivity of somatosensory cortical neurons of rabbits to acetylcholine and noradrenalin was investigated by microiontophoresis. Prostaglandin F₂ predominantly inhibited, whereas E₂ increased the spike activity of the neurons. F₂ usually modified the sensitivity of the neurons to acetylcholine, E₂ to noradrenalin. The influence of F₂ on the excitatory effects of acetylcholine, and of E₂ on the inhibitory effects of noradrenalin as a rule was characterized by weakening of the action of the mediators or a change in the sign of the initial responses. It is suggested that prostaglandins of the F group participate in cholinergic, and those of the E group in adrenergic transmission. The prostaglandins studied evidently exert their action through selective inhibition of the activity of adenylate or guanylate cyclases, leading to a decrease in the synthesis of secondary transmitters, namely cyclic AMP and cyclic GMP.B. K. Anokhin Research Institute of Normal Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 12, No. 3, pp. 239–245, May–June, 1980.     

Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis?

Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three biochemical properties of Bcl-2 proteins have been identified: their ability to localize constitutively and/or inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might control cell death through an ability to regulate mitochondrial physiology.     

Carbamylation of proteins in 2-D electrophoresis--myth or reality?

Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Detection of pro-apoptotic Bax∆2 proteins in the human cerebellum

Bax?2 is a pro-apoptotic protein originally discovered in colon cancer patients with high microsatellite instability. Unlike most pro-apoptotic Bax family members, Bax?2 mediates cell death through a non-mitochondrial caspase 8-dependent pathway. In the scope of analyzing the distribution of Bax?2 expression in human tissues, we examined a panel of human brain samples. Here, we report four cerebellar cases in which the subjects had no neurological disorder or disease documented. We found Bax?2 positive cells scattered in all areas of the cerebellum, but most strikingly concentrated in Purkinje cell bodies and dendrites. Two out the four subjects tested had strong Bax?2-positive staining in nearly all Purkinje cells; one was mainly negative; and one had various levels of positive staining within the same sample. Further genetic analysis of the Purkinje cell layer, collected by microdissection from two subjects, showed that the samples contained G7 and G9 Bax microsatellite mutations. Both subjects were young and had no diseases reported at the time of death. As the distribution of Bax?2 is consistent with that known for Baxα, but in a less ubiquitous manner, these results may imply a potential function of Bax?2 in Purkinje cells.     

Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure–function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b₅, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.     

Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.     

Modulation of endogenous prostaglandins by thymosin-α1 in lymphocytes

The effects of thymosin-α₁ on the stimulation of specific release of prostaglandin E₂ (PGE₂) from splenic lymphocytes and thymocytes were studied. Experiments were also performed to study in parallel the absolute levels of thymosin-α₁ in the blood and the induction of serum FTS activity and of azathioprine sensitivity of spleen cells from adult thymectomized (ATx) mice. A significant difference in the release of PGE₂ between normal splenocytes and splenocytes from ATx mice was observed. Thymosin-α₁ at certain concentrations was able to stimulate PGE₂ release from lymphocytes of ATx mice while inhibiting release in lymphocytes of normal mice. Also, thymocytes were stimulated to release PGE₂ after incubation with α₁ in a manner similar to that seen in spleen cells of ATx mice. Approximately the same concentration of α₁ was found to also correct the low azathioprine sensitivity of splenocytes from ATx mice. Determinations of FTS-like activity in the blood and the pharmacokinetics of α₁ after administration of this synthetic molecule show a clear dissociation. A maximum peak of α₁ activity was obtained after 1 hr, while maximal FTS-like activity was observed after 24 hr. The inhibition of the induction by α₁ of FTS-like activity and of Thy 1.2 antigen by indomethacin suggests that the action of α₁ requires prostaglandin biosynthesis.     

Classification of β-hairpin repeat proteins

In recent years, a number of new protein structures that possess tandem repeats have emerged. Many of these proteins are comprised of tandem arrays of β-hairpins. Today, the amount and variety of the data on these β-hairpin repeat (BHR) structures have reached a level that requires detailed analysis and further classification. In this paper, we classified the BHR proteins, compared structures, sequences of repeat motifs, functions and distribution across the major taxonomic kingdoms of life and within organisms. As a result, we identified six different BHR folds in tandem repeat proteins of Class III (elongated structures) and one BHR fold (up-and-down β-barrel) in Class IV (“closed” structures). Our survey reveals the high incidence of the BHR proteins among bacteria and viruses and their possible relationship to the structures of amyloid fibrils. It indicates that BHR folds will be an attractive target for future structural studies, especially in the context of age-related amyloidosis and emerging infectious diseases. This work allowed us to update the RepeatsDB database, which contains annotated tandem repeat protein structures and to construct sequence profiles based on BHR structural alignments.     

Decreases in the relative concentrations of specific hepatocyte plasma membrane proteins during liver regeneration: down-regulation or dilution?

Antibodies were used to quantify seven domain-specific integral proteins of the rat hepatocyte plasma membrane during rat liver regeneration in response to two-thirds hepatectomy. Quantitative immunoblotting revealed that a subset of the plasma membrane proteins exhibited transient 30-70% decreases in relative concentration during the period of hepatocyte proliferation. The list of affected proteins included at least one representative from each of the plasma membrane domains: the apical protein HA 4, the lateral protein HA 321, and the basolateral receptors for epidermal growth factor and asialoglycoproteins. In contrast, the relative concentrations of three other plasma membrane proteins, the basolateral protein CE 9 and the two apical proteins dipeptidylpeptidase IV and aminopeptidase N, remained unchanged throughout liver regeneration. The decreases in the relative concentrations of the plasma membrane proteins were observed even when the synthesis of hepatocyte DNA was blocked by hydroxyurea, suggesting that the signalling for these two delayed consequences of two-thirds hepatectomy occurred along parallel, dependent pathways. Pulse and pulse-chase metabolic radiolabeling studies revealed that the decreases in the concentrations of the PM proteins were accomplished through protein-selective decreases in the rates of synthesis of the high-mannose precursors of the affected proteins, but not through the accelerated degradation of the mature plasma membrane proteins.     

Effect of prostaglandins on α-aminoisobutylic acid uptake in cultured fibroblasts

Effect of various prostaglandins on the uptake of α-aminoisobutylic acid by cultured fibroblasts was studied. All the prostaglandins having an OH functional group in an intramolecular 5-membered ring showed an inhibitory effect on the amino acid uptake. The active compounds can be ranked in potency according to the values for the inhibition of the amino acid uptake per cent of control: prostaglandin F_2α(53 %) >F_1α(54 %) >D₂(56 %) >E₂(62 %) >thromboxane B₂ (66 %). Thus, prostaglandin F_2α was found to be the most potent inhibitor to membrane permeability and the inhibitory effect was dose dependent. The inhibition was maximal after 1 hour of exposure to prostaglandin F_2α, persisted at least up to 6 hours in the presence of prostaglandin F_2α.     

Are prostaglandins involved in atrial natriuretic peptide mechanisms of cardiovascular control?

Atrial natriuretic peptide (ANP) can excite cardiac nerve endings and invoke a decrease in arterial blood pressure and a reduction in renal sympathetic nerve activity. Our laboratory has previously demonstrated that this renal depressor reflex was invoked by systemic injection of ANP and not by the direct application of ANP to the epicardium, a major locus for vagal afferents. We now examine whether inhibition of prostaglandin synthesis impairs reflex responses that are normally associated with ANP injections. Renal sympathetic nerve activity, arterial blood pressure, and heart rate were recorded in anesthetized rats. Indomethacin was used to inhibit prostaglandin synthesis through the cyclooxygenase pathway. The ANP-mediated decrease in arterial blood pressure and renal sympathetic nerve activity, observed when prostaglandin synthesis was inhibited, did not differ significantly from the decreases observed in these parameters when prostaglandin synthesis was not inhibited. Heart rate remained unchanged. Our results suggest that the sympatho-inhibitory effects of ANP do not require prostaglandins as intermediary compounds.