Improved immunodetection of endogenous α-synuclein

α-Synuclein is a key molecule in understanding the pathogenesis of neurodegenerative α-synucleinopathies such as Parkinson's disease. Despite extensive research, however, its precise function remains unclear partly because of a difficulty in immunoblotting detection of endogenous α-synuclein. This difficulty has largely restricted the progress for α-synucleinopathy research. Here, we report that α-synuclein monomers tend to easily detach from blotted membranes, resulting in no or very poor detection. To prevent this detachment, a mild fixation of blotted membranes with paraformaldehyde was applied to the immunoblotting method. Amazingly, this fixation led to clear and strong detection of endogenous α-synuclein, which has been undetectable by a conventional immunoblotting method. Specifically, we were able to detect endogenous α-synuclein in various human cell lines, including SH-SY5Y, HEK293, HL60, HeLa, K562, A375, and Daoy, and a mouse cell line B16 as well as in several mouse tissues such as the spleen and kidney. Moreover, it should be noted that we could clearly detect endogenous α-synuclein phosphorylated at Ser-129 in several human cell lines. Thus, in some tissues and cultured cells, endogenous α-synuclein becomes easily detectable by simply fixing the blotted membranes. This improved immunoblotting method will allow us to detect previously undetectable endogenous α-synuclein, thereby facilitating α-synuclein research.     

Detection of endogenous β-glucuronidase activity in Aspergillus niger

 An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH. Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995     

Do T cells need endogenous peptides for activation?

T cells are sensitive to small numbers of antigenic peptide-MHC ligands that are distributed among an excess of endogenous peptide-MHC complexes on the surface of antigen-presenting cells. Although there are accumulating data that indicate a role for these endogenous peptide-MHC complexes in T-cell receptor triggering, whether they are necessary, and the nature of their function, is controversial. In this Opinion article, I argue that endogenous peptide-MHC complexes are required for T-cell stimulation and that their mechanism of action differs between CD4(+) and CD8(+) T cells.     

Do endogenous cannabinoids contribute to HIV-mediated immune failure?

The failure of the immune system to mount a successful attack on the human immunodeficiency virus (HIV) is an old enigma for AIDS research. The high mutational capacity of HIV, which unremittingly confuses the immune system, is a major factor in immune failure. But this alone cannot fully explain the certain and inescapable failure of the immune system, leading to full-blown AIDS. Here, we propose the hypothesis that endogenous cannabinoids, derived mostly from macrophages, might participate in the general failure of the immune system in HIV-infected individuals.     

Are endogenous clustered DNA damages induced in human cells?

Although clustered DNA damages are induced in cells by ionizing radiation and can be induced artifactually during DNA isolation, it was not known if they are formed in unirradiated cells by normal oxidative metabolism. Using high-sensitivity methods of quantitative gel electrophoresis, electronic imaging, and number average length analysis, we found that two radiosensitive human cell lines (TK6 and WI-L2-NS) accumulated Fpg-oxidized purine clusters and Nth-oxidized pyrimidine clusters but not Nfo-abasic clusters. However, four repair-proficient human lines (MOLT 4, HL-60, WTK1, and 28SC) did not contain significant levels (<5/Gbp) of any cluster type. Cluster levels were independent of p53 status. Measurement of glycosylase levels in 28SC, TK6, and WI-L2-NS cells suggested that depressed hOGG1 and hNth activities in TK6 and WI-L2-NS could be related to oxybase cluster accumulation. Thus, individuals with DNA repair enzyme deficiencies could accumulate potentially cytotoxic and mutagenic clustered DNA damages. The absence of Nfo-detected endogenous clusters in any cells examined suggests that abasic clusters could be a signature of cellular ionizing radiation exposure.     

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.     

SHARPIN is an endogenous inhibitor of β1-integrin activation

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.     

Do nitric oxide donors mimic endogenous NO-related response in plants?

Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 μM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that “donors are compounds that spontaneously break down to release NO” and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.     

The IAP family：endogenous caspase inhibitors with multiple biological activities

IAPs (inhibitors of apoptosis) are a family of proteins containing one or more characteristic BIR domains.These proteins have multiple biological activities that include binding and inhibiting caspases,regulating cell cycle progression,and modulating receptor-mediated signal transduction.Our recent studies found the IAP family members XIAP and c-IAP1 are ubiquitinated and degraded in proteasomes in response to apoptotic stimuli in T cells,and their degradation appears to be important for T cells to commit to death.In addition to three BIR domains,each of these IAPs also contains a RING finger domain. We found this region confers ubiquitin protease ligase(E3) activity to IAPs,and is responsible for the auto-ubiquitination and degradation of IAPs after an apoptotic stimulus.Given the fact that IAPs can bind a variety of proteins,such as caspases and TRAFs,it will be of interest to characterize potential substrates of the E3 activity of IAPs and the effects of ubiquitination by IAPs on signal transduction,cell cycle,and apoptosis.     

Distribution of endogenous and exogenous 5′-nucleotidase on bovine spermatozoa

A polyclonal rabbit antibody against 5-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.     

Glycogen phosphorylase ‘b’ in Dictyostelium: stability and endogenous phosphorylation

Summary The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase b form requires 5 AMP for activity and is present in early development. The active phosphorylase a form is 5 AMP independent and occurs during later development. We here show that the 92 kd b enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn²⁺ and ATP, the phosphorylase b shows apparent conversion into a 5 AMP independent form as measured by enzyme activity. In addition, Mn²⁺ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase b subunit. We also demonstrate phosphorylation of the b enzyme subunit in vivo by ³²-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase b subunit is characterized.     

Are human endogenous retroviruses pathogenic? An approach to testing the hypothesis

A number of observations have led researchers to postulate that, despite being replication‐defective, human endogenous retroviruses (HERVs) may have retained the potential to cause or contribute to disease. However, mechanisms of HERV pathogenicity might differ substantially from those of modern infectious retroviruses or of the infectious precursors of HERVs. Therefore, novel pathways of HERV involvement in disease pathogenesis should be investigated. Recent technological advances in sequencing and bioinformatics are making this task increasingly feasible. The accumulating knowledge of HERV biology may also facilitate the definition and general acceptance of criteria that establish HERV pathogenicity. Here, we explore possible mechanisms whereby HERVs may cause disease and examine the evidence that either has been or should be obtained in order to decisively address the pathogenic potential of HERVs.     

Neuroprotective–neurotrophic effect of endogenous dehydroepiandrosterone sulfate during intense stress exposure

Recent reports demonstrate neurotrophic properties of dehydroepiandrosterone sulfate (DHEAS) in men at rest, as well as profound neurotrophic responses to stress in both men and women. Little is known of neuroprotective–neurotrophic effects of DHEAS during stress exposure, either in men or women. This translational study was designed to examine neuroprotective–neurotrophic effects of DHEAS throughout intense stress exposure in healthy men and women. The study took place within a stressful 12-day military survival course. Utilizing a longitudinal cross-sectional repeated measures design, One hundred sixteen healthy active-duty military personnel (80% male) were studied before, during, and 24 h after the course. The dependent variable was the neurotrophin salivary nerve growth factor (sNGF). In terms of total hormone output, the effect of DHEAS on sNGF was mediated by testosterone. Unlike testosterone or cortisol, DHEAS reliably predicted sNGF at each time point, and change in DHEAS predicted change in sNGF across time points. Baseline DHEAS predicted total sNGF output across the stress trajectory. Consistent with preclinical as well as cross-sectional human research, this study demonstrates neuroprotective–neurotrophic effects of DHEAS in healthy men and women exposed to intense stress. Results are evaluated in relation to established criteria for causation.     

Do panther chameleons bask to regulate endogenous vitamin D3 production?

Basking by ectothermic vertebrates is thought to have evolved for thermoregulation. However, another beneficial effect of sunlight exposure, specifically the ultraviolet B (UV-B) component, includes endogenous production of vitamin D(3). In the laboratory, panther chameleons exhibited a positive phototaxis to greater visible, ultraviolet A (UV-A) and UV-B light. However, with equivalent high irradiances of UV-A or UV-B, their response to UV-B was significantly greater than it was to UV-A. Exposure of in vitro skin patches of panther chameleons to high UV-B (90 microW/cm(2)) for 1 h significantly enhanced vitamin D(3) concentration. Voluntary exposure to higher UV-B irradiance (70 vs. 1 microW/cm(2)) resulted in greater circulating 25-hydroxyvitamin D(3) in female panther chameleons (604 vs. 92 ng/mL). Depending on dietary intake of vitamin D(3), chameleons adjusted their exposure time to UV-B irradiation as if regulating their endogenous production of this vital hormone. When dietary intake was low (1-3 IU/g), they exposed themselves to significantly more UV-producing light; when intake was high (9-129 IU/g), they exposed themselves to less. Vitamin D(3) photoregulation seems to be an important additional component of the function of basking.     

Receptor desensitization by neurotransmitters in membranes: are neurotransmitters the endogenous anesthetics?

A mechanism of anesthesia is proposed that addresses one of the most troubling peculiarities of general anesthesia: the remarkably small variability of sensitivity within the human population and across a broad range of animal phyla. It is hypothesized that in addition to the rapid, saturable binding of a neurotransmitter to its receptor that results in activation, the neurotransmitter also acts indirectly on the receptor by diffusing into the postsynaptic membrane and changing its physical properties, causing a shift in receptor conformational equilibrium (desensitization). Unlike binding, this slower indirect mechanism is nonspecific: each neurotransmitter will, in principle, affect all receptors in the membrane. For proteins modeled as having only resting and active conformational states, time-dependent ion currents are predicted that exhibit many characteristics of desensitization for both inhibitory and excitatory channels. If receptors have been engineered to regulate the time course of ion currents by this mechanism, then (a) mutations that significantly alter receptor sensitivity to this effect would be lethal and (b) by design, excitatory receptors would be inhibited, but inhibitory receptors activated, so that their effects are not counterproductive. The wide range of exogenous molecules that affect the physical properties of membranes as do neurotransmitters, but that do not bind to receptors, would thus inhibit excitatory channels and activate inhibitory channels, i.e., they would act as anesthesics. The endogenous anesthetics would thus be the neurotransmitters, the survival advantage conferred by their proper membrane-mediated desensitization of receptors explaining the selection pressure for anesthesic sensitivity.     

Polymorphism and chromosomal location of endogenous α-amylase inhibitor genes in common wheat

Summary Polymorphism of an endogenous -amylase inhibitor in wheat was studied using iso-electric focusing followed by monoclonal antibody — based immunoblotting. Ten isoforms of the inhibitor detected in common wheat and its wild counterparts were assigned to five homoeologous loci. Three -amylase inhibitor loci (Isa-1) were identified in common wheat and located on the long arms of chromosomes 2A, 2B and 2D. In a sample of 27 bread wheats, eight durum wheats, and 12 diploid wheat relatives, amphiploids and triticales, a high resolution isoelectric-focusing separation demonstrated two active and one null allele at the Isa-A1, two alleles at the Isa-B1, one allele at the Isa-D1, four alleles at the Isa-S1, and one allele at the Isa-G1 locus. The most frequent electrophoretic pattern of common wheat cultivars consisted of two isoforms, encoded respectively by the Isa-B1b, Isa-D1 a alleles and the Isa-Alnull allele. All the durum wheats had only one inhibitor form controlled by allele Isa-B1b, which was accompanied by the null allele at the Isa-A1 locus.Contribution No. 210 of the Food Science Department, University of Manitoba     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.