Structure-specific binding recognition of a methanogen chromosomal protein

BACKGROUND: Liver dysfunction may be an early event or the end result of multiple organ dysfunction (MOD) in necrotizing pancreatitis. This study measured the early changes in hepatocellular ions and energetics associated with such conditions. METHODS: Twenty-five rats, prepared with a 23Na and 31P double-tuned nuclear magnetic resonance surface coil secured over the dome of the liver, were randomized into 5 groups: control, 10 and 20 minutes of total inflow ischemia, pancreatitis induced by deoxycholic acid (DCA), and sham-DCA (saline injection). Dysprosium-TTHA3- solution was used to separate the intracellular and extracellular sodium peaks. RESULTS: In rat liver, 20 minutes of total inflow occlusion caused irreversible depletion of high-energy phosphates. Changes at 2 hours after the onset of DCA-pancreatitis are compared with changes after 20 minutes of ischemia (mean +/- SEM). Although the DCA-pancreatitis animals did not become hypotensive until 1 hour after the induction of pancreatitis, the changes in hepatic intracellular ions and energetics began soon after such an insult. At 2 hours after the onset of pancreatitis, hepatocellular pHi and [NA+]i were 6.99 +/- 0.16 and 78.4 +/- mmol/L, respectively (P < .01, compared with sham animals). A similar pattern of changes in hepatic bioenergetics also occurred. After the onset of pancreatitis, the hepatic cytostolic phosphorylation potential decreased with time (y = 0.654 - 0.004t, where t is time in minutes and r2 = 0.967 and the rate of hepatic hydrolysis of adenosine triphosphate increased progressively (y = 0.702t + 91.363, where t is time in minutes and r2 = 0.969. These changes correlated well with the accumulated [Na]i. CONCLUSIONS: Unresuscitated necrotizing pancreatitis caused severe hepatocellular acidosis, profound sodium accumulation, and bioenergy depletion early in its course. These effects were as severe as those induced by total liver ischemia. Liver dysfunction may be an early, not terminal, event of MOD in necrotizing pancreatitis.     

The giant protein titin. Emerging roles in physiology and pathophysiology

Activation of the nociceptin receptor stably expressed in Chinese hamster ovary cells induced a transient mitogen-activated protein kinase (MAPK) activation, via pertussis toxin-sensitive G-proteins. The nociceptin receptor-mediated MAPK activation was partially blocked by down-regulation or inhibition of protein kinase C, and suppressed by pretreatment with a phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, a tyrosine protein kinase inhibitor, genistein, and phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, affected the nociceptin-induced MAPK activity. The nociceptin-induced MAPK activation may lead to activation of phospholipase A2 and induce changes in gene expression.     

Production of the thyrotrophin receptor extracellular domain as a glycosylphosphatidylinositol-anchored membrane protein and its interaction with thyrotrophin and autoantibodies

The thyrotrophin (TSH) receptor (TSHR) is synthesized as a single polypeptide with a predicted large extracellular domain (ECD), a seven-transmembrane pass region and a C-terminal intracellular tail. It is a common target for production of autoantibodies. To investigate whether the ECD is solely responsible for ligand interaction, we directed the expression of this domain in isolation on the cell surface by means of a glycosylphosphatidylinositol (GPI) anchor sequence. Immunoblotting detected TSHR material of Mr 70,000 expressed at high levels. In immunoprecipitation studies, the GPI-anchored ECD was recognized by experimental and pathological antibodies. The molecule was detected on the cell surface by flow cytofluorimetry at up to 10-fold higher amounts than the highest expressing full-length receptor clone. Radioligand binding studies confirmed this and showed that the recombinant molecule bound TSH with high affinity similar to full-length receptor; however, studies with human autoimmune sera indicated differences in the degree of inhibition when compared with full-length receptor. The existence of the GPI anchor was confirmed by cleavage with a GPI-specific phospholipase C and biosynthetic labeling with [3H]ethanolamine. TSHR material was also present inside the cell in both soluble and membrane-bound forms. Thus, the recombinant GPI-anchored ECD is the smallest known fragment of the TSHR that retains high-affinity TSH binding and is expressed at high levels on the cell surface as well as internally; this approach may well be useful for other membrane proteins.     

Human H-kininogen is a ferritin-binding protein

H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.     

Yeast Ku as a regulator of chromosomal DNA end structure

During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand. Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle. In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA. These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by functioning as a terminus-binding factor. Because Ku-mediated DNA end joining involving telomeres would result in chromosome instability, our data also suggest that Ku has a distinct function when bound to telomeres.     

Increased nuchal translucency as a marker for fetal chromosomal defects

BACKGROUND: Screening for trisomy 21 (Down's syndrome) by measuring maternal serum alpha-fetoprotein, chorionic gonadotropin, and estriol concentrations and then performing chorionic-villus sampling or amniocentesis identifies approximately 60 percent of fetuses with this disorder. We used ultrasonography to detect increased nuchal translucency and cystic hygroma, which are characteristic features of fetuses with chromosomal defects. METHODS: We performed transvaginal ultrasonography in 10,010 unselected adolescents and women less than 40 years of age with live singleton fetuses at 10 to 15.9 weeks of gestation. Increased fetal nuchal translucency was defined as an area of translucency at least 3 mm in width, and cystic hygromas were defined as septated, fluid-filled sacs in the nuchal region. Subjects whose fetuses had these findings were offered fetal karyotyping. Information on pregnancies, deliveries, and neonates was subsequently obtained from hospital records and national birth and malformation registries. RESULTS: Nuchal translucency or cystic hygroma was seen in 76 fetuses (0.8 percent), of which 18 (24 percent) had an abnormal karyotype. The sensitivity for trisomies 21, 18, and 13 combined was 62 percent (13 of 21 fetuses), and the sensitivity for trisomy 21 alone was 54 percent (7 of 13 fetuses). CONCLUSIONS: The use of transvaginal ultrasonography to detect increased nuchal translucency and cystic hygroma is a sensitive test for fetal aneuploidy. It can be done earlier in pregnancy than serum screening, and it decreases the subsequent need for chorionic-villus sampling or amniocentesis.     

Human dentine as a hydrogel

The pores (tubules) of human dentine in 0.02-cm planoparallel sections of newly extracted permanent teeth were investigated. By the conventional scanning electron microscopy these pores appear empty, but by the newly developed scanning-probe microscopy the presence of a complex matrix could be established. By measuring the transport of neutral myoglobin by diffusion alone and diffusion+bulk flow, the area of dentine occupied by the matrix was calculated to be 1.9 +/- 0.9% and 2.3 +/- 0.5%, respectively. The hydraulic conductivity was surprisingly small, 1.35 +/- 0.55 x 10(-7) ml/(s.cm2 dentine) at a pressure difference of 0.1 kPa across a 1-cm thick section. This suggests a hydrogel with a relatively dense network, the width of meshes estimated at 2 x 30 nm. In line with this concept, enzymatic degradation of the organic matter increased the hydraulic conductivity 3000 times. By studying the transport of negatively charged myoglobin, the matrix was calculated to carry 18 mEq/l of positive charges. Due to the consequent attraction of small, negative ions and thence of water, the pressure within the matrix would be about 1.33 kPa, a force which will act to immobilize the water in the channels. The concept of a hydrogel in the dentine tubules was also supported by the finding that shielding the charges with bathing media of high ionic strength reduced the hydraulic conductivity.     

Human serum albumin as a probe for protein adsorption to nanoparticles: relevance to biodistribution

A range of poloxamers and poloxamines were adsorbed to biodegradable poly(lactide-co-glycolide) (PLGA) and non-biodegradable polystyrene (PS) particulate systems in order to alter their surface characteristics and produce potential drug targeting systems. Human serum albumin (HSA) was chosen as a model protein to investigate protein adsorption to the above systems and was quantified by two techniques. I125 radiolabelled HSA proved to be a useful probe for determining protein adsorption but was limited by a modification that occurred on storage. Also, HSA eluted from the particle surface was quantified by densitometry following it's development on an SDS-PAGE gel. Both techniques produced similar results. For cleaned coated PS particles it was found that the PEO chain length and the molecular structure of the block copolymer were important in preventing protein adsorption. The presence of excess block copolymer in the uncleaned preparations resulted in further suppression of HSA adsorption, which was thought to be due to their detergent properties. Due to the different results obtained with similarly coated PLGA particles, it was concluded that the block copolymers adsorb onto the surface of the PLGA particles in a different conformation to those adsorbed onto PS particles. Correlating in vivo biodistribution in terms of the prevention of protein (opsonin) adsorption was of only limited success and it was concluded that adsorption data for a single model protein can only be used with caution to predict the in vivo behaviour of colloidal targeting systems.     

Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency

This study was undertaken to analyze antibodies to protein S (PS) in patients with an acquired PS deficiency. Plasma from symptomatic patients with acquired (n = 14) or congenital (n = 10) PS deficiency and 10 healthy donors was screened for PS antibodies by immunoblotting and for anti-phospholipid antibodies. PS antibodies (IgG) were detected in five of the patients with acquired PS deficiency. These antibodies belonged to the G1 and G4 immunoglobulin subclasses. IgG fractions from the same 5 patients were shown to inhibit PS activity. The inhibition of PS activity by the 5 IgG fractions was shown to be time- and dose-dependent and was abolished following incubation with purified PS, while no effect was found after absorption with cardiolipin micelles. In addition, anticardiolipin monoclonal or human purified antibodies, failed to exert significant PS inhibition. These findings demonstrate that anti-PS antibodies are able to inhibit PS activity and that this is independent of anti-phospholipid antibodies. Given the clinical features of the patients, these antibodies should be regarded as an expression of the broad autoimmune syndrome involving the phospholipid-binding plasma proteins.     

HMG-17, a chromosomal non-histone protein, shows developmental regulation during organogenesis

A significant portion of patients who present with non-muscle invasive "superficial" bladder cancer develop the muscle "invasive" life-threatening form of the disease during subsequent follow-up. In clinical studies, overexpression of the epidermal growth factor receptor (EGFR) and the p21 ras oncogene have been strongly associated with this phenotypic tumor transition. The marked difference in incidence of invasive bladder cancer in Asia compared to the United States has made us hypothesize that, among other factors, dietary influences have an impact on such tumor progression. A significantly higher dietary consumption of soy products exists in Asia and has led to the notion that the isoflavones present in this diet may contribute to a reduction in the number of invasive transitional cell bladder cancers. In this regard, we sought to determine the effect of genistein, a naturally occurring dietary protein tyrosine kinase (PTK) inhibitor, on the growth and motility of human bladder cancer cell lines with diverse EGFR and p21ras expression phenotypes and corresponding invasive behaviors. These effects were compared with those of tyrphostin, a pure synthetic EGFR inhibitor. Our results indicate that both genistein and tyrphostin are effective inhibitors of bladder cancer motility and growth, key factors in the development of muscle invasive disease. In addition, the growth and motility inhibitory effects of genistein and tyrphostin are observed preferentially in cells that overexpress the EGFR. Cells that have a mutated p21ras but do not overexpress the EGFR are less inhibited by these 2 compounds, suggesting that their effect is primarily directed at the EGFR signal transduction pathways proximal to the p21ras gene. Our results would seem to corroborate the notion that a high dietary intake of isoflavones is a likely explanation for the decreased incidence of invasive bladder cancer.     

Human subconscious as a p-adic dynamical system

We propose a mathematical model of the process of thinking based on dynamical systems over a configuration space of ideas. These dynamical systems are assumed to be located in the human subconscious and are controlled by the human conscious which fixes parameters of the dynamical systems in the subconscious and transmits to the subconscious generating ideas which initiate iterations of the dynamical systems in the subconscious. Thus, we present the mathematical model which is not based on the rule of reason. Mathematically the space of ideas is described by so-called p-adic numbers. In fact, a p-adic metric on the space of ideas corresponds to the following nearness between ideas: two ideas x and y are close if they have sufficiently long common root. Already the simplest p-adic dynamical systems might describe some sides of human psychological and social behaviour.     

Human anti-Ro autoantibodies bind multiple conformational epitopes of 60-kD Ro autoantigen

Bronchial asthma is characterized by eosinophil infiltration and tissue remodeling. Matrix metalloproteinases (MMPs) are thought to play critical roles by degradating interstitial matrices in a wide range of lung diseases associated with reorganization of the airway architecture. To investigate whether MMPs are involved in the pathologic processes of bronchial asthma, we examined MMP expression in asthmatic subjects. In situ hybridization revealed abundant expression of MMP-9 (gelatinase B) mRNA in biopsy specimens from asthmatic subjects (n = 5), with an average positive cell distribution of 117.8 +/- 41.1 (mean +/- SEM)/mm2. In contrast, sparse expression of the mRNA (10.8 +/- 4.8 /mm2) was observed in specimens from normal subjects (n = 4). The vast majority of cells expressing the mRNA were eosinophils in asthmatic tissues (92.2 +/- 1.2%). MMP-9 protein, which was confined to the submucosal cells in the normal subjects, was not abundantly expressed in inflammatory cells, but there was positive reactivity for MMP-9 protein in the extracellular matrix. Immunoelectron microscopic analysis showed sparse immunolocalization of MMP-9 in the perinuclear spaces of eosinophils, but not in the granules. These findings suggest the overexpression of MMP-9 by eosinophils in bronchial tissues of asthmatic individuals, and the participation of MMPs in the pathologic changes in asthmatic airways.     

Human amniotic IgA inhibits natural IgG autoantibodies of maternal or unrelated origin

We show that the natural autoantibody activity of amniotic IgG dramatically increases after purification, and that the IgG-depleted fraction can suppress the activity of IgG natural antibodies from amniotic fluid or from the maternal serum. This suppression is also observed towards serum IgG from unrelated adults but does not impair the tetanus antitoxin activity of serum-derived IgG. Absorption experiments and immunoglobulin separation by gel permeation demonstrate that this suppression is due to monomeric immunoglobulins of the IgA isotype. The inhibition is associated with an anti-F(ab')2 activity of the amniotic IgA, involving hypervariable regions of the IgG as demonstrated by different reactivities towards monoclonal IgG sharing the same family of VH and Vkappa domains. These results indicate that the inhibition of natural autoantibodies not only occurs with fetal and adult serum IgM, as reported by other groups, but also with amniotic IgA, suggesting a general and important phenomenon. In the case of the amniotic fluid, IgA could protect the fetus against maternal IgG autoantibodies without interfering with simultaneously translocated antigen-induced IgG antibodies to pathogens.     

Human La protein: a stabilizer of histone mRNA

Histone mRNA is destabilized at the end of S phase and in cell-free mRNA decay reaction mixtures supplemented with histone proteins, indicating that histones might autoregulate the histone mRNA half-life. Histone mRNA destabilization in vitro requires three components: polysomes, histones, and postpolysomal supernatant (S130). Polysomes are the source of the mRNA and mRNA-degrading enzymes. To investigate the role of the S130 in autoregulation, crude S130 was fractionated by histone-agarose affinity chromatography. Two separate activities affecting the histone mRNA half-life were detected. The histone-agarose-bound fraction contained a histone mRNA destabilizer that was activated by histone proteins; the unbound fraction contained a histone mRNA stabilizer. Further chromatographic fractionation of unbound material revealed only a single protein stabilizer, which was purified to homogeneity, partially sequenced, and found to be La, a well-characterized RNA-binding protein. When purified La was added to reaction mixtures containing polysomes, a histone mRNA decay intermediate was stabilized. This intermediate corresponded to histone mRNA lacking 12 nucleotides from its 3' end and containing an intact coding region. Anti-La antibody blocked the stabilization effect. La had little or no effect on several other cell cycle-regulated mRNAs. We suggest that La prolongs the histone mRNA half-life during S phase and thereby increases histone protein production.     

cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients

There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.