The acute phase response in apolipoprotein A-1 knockout mice: apolipoprotein serum amyloid A and lipid distribution in plasma high density lipoproteins

In plasma, the bulk of apoSAA, a positive acute phase reactant protein, is transported in high density lipoproteins (HDL), especially HDLH (apoA1-rich HDL). In this study we tested whether apoA1 deficiency would adversely affect apoSAA concentration and lipid distribution in mouse plasma lipoproteins. Acute phase response (APR) was induced in C57BL/6J (apoA1+/+) and apoA1-knockout mice (apoA1-/-) by a subcutaneous injection of silver nitrate. The APR increased cholesterol concentrations in LDL of apoA1-/- mice and apoA1+/+ mice in a like manner. In contrast to apoA1+/+ mice, concentrations of cholesterol, phospholipids and proteins in both HDLL (1.063相似文献   

Purification of recombinant apolipoprotein A-1Milano expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation

Immunohistochemistry using anti-human neuron-specific enolase (NSE) mouse monoclonal antibody was performed in human brains from autopsy cases, which enabled us to assess the neuronal damage besides hematoxylin and eosin or Klüver-Barrera stain. Neurons in cerebral neocortex which showed necrotic changes such as prominent cytoplasmic vacuolization or cellular shrinkage with nuclear pyknosis showed a tendency to be less stained by anti-NSE antibody. Anti-NSE immunostaining was statistically significantly less in the neocortex from CO intoxication than from other causes of death, although morphological necrotic changes were less observed in CO intoxication. Hippocampal CA1 neurons clearly lost NSE immunoreactivity with the progression of necrotic changes. Neurons in CA2 were statistically significantly better stained by anti-NSE antibody than in CA1, 3, and 4. Cerebellar Purkinje cells were poorly stained by anti-NSE antibody, whereas neurons in cerebellar dentate nucleus and inferior olive in medulla oblongata were better stained. Anti-NSE immunostaining was lost in the injured areas of the cerebral neocortex while neurons in the intact areas were better stained in brain injury. These results indicate that anti-NSE immunostaining of neurons could reflect vital reaction and could be useful in evaluating neuronal damage in the hippocampal CA1 region or brain injury.     

Primary structure of chicken cardiac myosin S-1 heavy chain

The sequence of the NH2-terminal 830 amino acid residues of chicken cardiac ventricular muscle myosin subfragment-1 (S-1) was determined. S-1 was obtained by limited chymotryptic digestion, and cleaved into three characteristics fragments (23, 41, and 22 kDa fragments) by limited tryptic digestion. These fragments were isolated by gel filtration on a Sephadex G-100 column, followed by cation-exchange chromatography on a CM-52 column and reverse-phase HPLC. The isolated fragments were sequenced completely. Peptides overlapping the 23 and 41 kDa fragments and also overlapping the 41 and 22 kDa fragments were obtained by cleaving S-1 with cyanogen bromide, and sequenced completely. We also obtained a minor fragment, the 20 kDa fragment, in addition to the three characteristic fragments. Amino acid compositions of the cyanogen bromide peptides of the 20 kDa fragment indicated that a portion of S-1 heavy chains had lost their COOH-terminal 21 residues during limited tryptic digestion. Methylated amino acid residues were found at four positions: epsilon-N-monomethyllysine at position 32, epsilon-N-trimethyllysine residues at 127 and 549, and 3-N-methylhistidine at 754.     

Extensive intimal apolipoprotein A1-derived amyloid deposits in a patient with an apolipoprotein A1 mutation

With the aim of identifying molecules that are expressed specifically in the brain during neurogenesis, we tried to generate monoclonal antibodies which recognize molecules showing unique temporal expression patterns and molecular characteristics. We used a homogenate of the rat fetal forebrain (day 12 of fetal life, E12) as an immunogen, and antibodies which reacted with this preparation were screened by immunoblotting. One of the antibodies, Mab3C8, recognized a 100-kDa antigen that is enriched in fetal brain. This 100-kDa antigen was constantly expressed during fetal life (from E12 to E20) and became scarcely detectable two days after birth. The antigen was detected in the insoluble fraction of fetal brain and its isoelectric point ranged from 6 to 7, suggesting that it was a membrane-coupled glycoprotein. Analysis by glycosidase treatment and lectin blotting suggested that it was an O-linked glycoprotein with an alpha2,6 sialyl linkage. Thus, a molecule unique to the fetal brain, an O-linked sialoglycoprotein with a molecular mass of 100 kDa (FOG100), was found by generating an antibody.     

Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks

In reconstituted high-density lipoproteins, apolipoprotein A-I and phosphatidylcholines combine to form disks in which the amphipathic alpha-helices of apolipoprotein A-1 bind to the edge of a lipid bilayer core, shielding the hydrophic lipid tails from the aqueous environment. We have employed experimental data, sequence analysis, and molecular modeling to construct an atomic model of such a reconstituted high-density lipoprotein disk consisting of two apolipoprotein A-I proteins and 160 palmitoyloleoylphosphatidylcholine lipids. The initial globular domain (1-47) of apolipoprotein A-I was excluded from the model, which was hydrated with an 8-A shell of water molecules. Molecular dynamics and simulated annealing were used to test the stability of the model. Both head-to-tail and head-to-head forms of a reconstituted high-density lipoprotein were simulated. In our simulations the protein contained and adhered to the lipid bilayer while providing good coverage of the lipid tails.     

Primary structure of bovine adenosine deaminase

Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.     

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.     

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.     

Primary structure of Streptomyces griseus metalloendopeptidase II

Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous "serine protease inhibitors" produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a "His-Glu-Xaa-His" motif of SGMPII was identified as the residue modified by CICH2 CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.     

Lipoprotein (a) and apolipoprotein A-1 and B in schoolchildren whose grandparents had coronary and cerebrovascular events: a preliminary study of 12-13 year old Japanese children

The aim of this study was to evaluate the relationship between the serum levels of lipoprotein (a) [Lp (a)] and apolipoproteins (apo A-1 and apo B) in schoolchildren with a history of coronary and cerebrovascular events in their grandparents. We measured serum concentrations of Lp (a) and apoliproteins immunochemically in 289 schoolchildren aged 12-13 years and questioned parents about coronary and cerebrovascular events in the children's grandparents. In boys and girls, mean +/- s.d. levels of apo A-1, apo B and Lp (a) were 134 +/- 20.3 and 136 +/- 17.4 mg/dL, 61 +/- 16 and 66 +/- 15 mg/dL and 12.5 +/- 15.3 and 12.5 +/- 15.1 mg/dL, respectively. There were no significant sex differences in the levels of apo A-1, apo B, and Lp (a). The Lp (a) levels (mean +/- s.d., 12.5 +/- 15.2 mg/dL; median 7.5 mg/dL, n = 289) were not affected by other variables. The Lp (a) distribution was strongly positively skewed and 75% of schoolchildren had very low levels. In the total 289 schoolchildren, thirty-two grandparents who had had coronary vascular events (21 myocardial infarction, 11 angina pectoris) and twenty-three grandparents who had had cerebrovascular events were recorded. By the boxplot statistical analysis, no difference was found in Lp (a) levels in children whose grandparents had myocardial infarction compared with those whose grandparents had no such history, or compared with those whose grandparents had suffered cerebrovascular events.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of the novel bacterial rhodopsin from extremely halophilic archaeon Haloarcula japonica strain TR-1

A novel bacterial rhodopsin was identified in Haloarcula japonica strain TR-1. The gene encoding the bacterial rhodopsin was cloned and sequenced. The structural gene consisted of an open reading frame of 750 nucleotides encoding 250 amino acids. The deduced amino acid sequence of the Ha. japonica bacterial rhodopsin showed the highest homology to those of cruxrhodopsins.     

Primary structure of trypsin inhibitors from Sicyos australis

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.     

Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The amino acid sequence of the p-hydroxybenzoate hydroxylase (4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) monomer from Pseudomonas fluorescens has been determined. The sequence was elucidated by a combination of the results from an X-ray crystallographic study at 0.25 nm resolution (Wierenga, R.K., de Jong, R.J., Kalk, K.H., Hol, W.G.J. and Drenth, J. (1979) J. Mol. Biol. 131, 55-73) and from protein sequence analysis. The polypeptide chain of the monomer contains 394 amino acids and has a molecular weight of 44 299.     

Noradrenaline contractions of human prostate mediated by alpha 1A-(alpha 1c-) adrenoceptor subtype

1. The subtype of alpha 1-adrenoceptor mediating contractions of human prostate to noradrenaline was characterized by use of a range of competitive and non-competitive antagonists. 2. Contractions of the prostate to either noradrenaline (pD2 5.5), phenylephrine (pD2 5.1) or methoxamine (pD2 4.4) were unaltered by the presence of neuronal and extraneuronal uptake blockers. Noradrenaline was about 3 and 10 times more potent than phenylephrine and methoxamine respectively. Phenylephrine and methoxamine were partial agonists. 3. Pretreatment with the alkylating agent, chlorethylclonidine (10(-4M) shifted the noradrenaline concentration-contraction curve about 3 fold to the right and depressed the maximum response by 31%. This shift is 100 fold less than that previously shown to be produced by chlorethylclonidine under the same conditions on alpha 1B-adrenoceptor-mediated contractions. 4. Cumulative concentration-contraction curves for noradrenaline were competitively antagonized by WB 4101 (pA2 9.0), 5-methyl-urapidil (pA2 8.6), phentolamine (pA2 7.6), benoxathian (pA2 8.5), spiperone (pA2 7.3), indoramin (pA2 8.2) and BMY 7378 (pA2 6.6). These values correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha 1c-adrenoceptors and poorly with values from cloned alpha 1b- and alpha 1d-adrenoceptors. 5. The good correlation between the functional data on the prostate and the binding data on the expressed alpha 1c-subtype clone for the affinities of the competitive antagonists suggests that they are the same subtype. As the expressed alpha 1c-adrenoceptor clone corresponds to the alpha 1A-adrenoceptor expressed in tissues, contraction of the human prostate to noradrenaline is therefore mediated by an alpha 1A-adrenoceptor.     

Hereditary amyloid cardiomyopathy caused by a variant apolipoprotein A1

Autosomal dominant hereditary amyloidosis with a unique cutaneous and cardiac presentation and death from heart failure by the sixth or seventh decade was found to be associated with a previously unreported point mutation (thymine to cytosine, nt 1389) in exon 4 of the apolipoprotein A1 (apoA1) gene. The predicted substitution of proline for leucine at amino acid position 90 was confirmed by structural analysis of amyloid protein isolated from cardiac deposits of amyloid. The subunit protein is composed exclusively of NH2-terminal fragments of the variant apoA1 with the longest ending at residue 94 in the wild-type sequence. Amyloid fibrils derived from four previously described apoA1 variants are composed of similar fragments with carboxyl-terminal heterogeneity, but contrary to those variants, which all carry one extra positive charge, the substitution Leu90Pro does not result in any charge modification. It is unlikely, therefore, that amyloid fibril formation is related to change of charge for a specific residue of the precursor protein. This is in agreement with studies on transthyretin amyloidosis in which no unifying factor such as change of charge for amino acid residues has been noted.     

Influence of macrophage-derived apolipoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apolipoprotein E-deficient mice

Two-dimensional multiple-quantum magic angle spinning (MQMAS) NMR and MAS NMR of 11B at various magnetic fields, were applied to elucidate the structure of vitreous (glassy) boron trioxide (v-B2O3), vitreous boron trisulfide (v-B2S3) and crystalline boron trisulfide (c-B2S3). These techniques, when combined with computer simulations of the resulting spectra, provide the isotropic chemical shifts and the quadrupole parameters, as well as a quantitative measure of the intensities of various boron resonances. The MAS NMR of v-B2O3 produced overlapping anisotropic lineshapes corresponding to the -1/2<-->1/2 transition in two distinct types of BO3 units with 3(+/-0.08):] intensity ratio. A combination of MAS and the multiple-quantum method resulted in a better resolved, isotropic 11B spectrum of v-B2O3. A remarkable enhancement of resolution of the MQMAS NMR proved instrumental in finding and identifying various impurities present in v-B2S3 and c-B2S3. In addition to the resonances from boron in two types of BS3 groups, four other structural units, BOS2, BO2S, BO3 and BS4, were elucidated from the spectra of vitreous and crystalline samples. The effects of various experimental parameters, such as the magnitude of the B0 and B1 fields, on the resolution of the MAS and MQMAS techniques are also shown.     

Selective enrichment with alpha 1A- and alpha 1B-adrenoceptor subtypes in rat brain cortical membranes

In this controlled, prospective and partially blind study two groups of patients with temporal lobe epilepsy were evaluated, respectively with good and bad prognosis. Measurements of epileptogenesis were based on frequency of seizures, and on epiletogenic electroencephalographic abnormalities obtained from scalp electrodes over the temporal lobes. The results were analysed by non-parametric analysis of variance, comparison of groups and analysis of correlation. The results indicated the temporal lobe groups were similar to at least one of the control groups in age, sex, educational and social level, therapeutic regime, age at onset and length of history of epilepsy. The quantitative measurements showed a global difference between the group of temporal lobe with bad and good prognosis, reaching statistical significance in clinical epileptogenesis, and a trend towards greater epileptogenesis on the electroencephalogram, in the same group of patients. The results indicate the experimental usefulness of some of the original measurements used in the study, but also their problems. A review of the literature is carried out.     

Association of phosphoinositide-specific phospholipase Cgamma1 with elements of cytoskeleton in A-431 cells

It is shown that phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1), a substrate of growth factor receptors, is associated with cytoskeleton in A-431 cells. PLC gamma 1 is co-localized only with the cortical actin but not with actin stress fibers. Since EGF receptor is also co-localized with the cortical actin it is concluded that PLC gamma 1 co-localized with actin is mediated by the EGF receptor. After the treatment with cytochalasin B PLC gamma 1 is co-localized with actin aggregates and cytoskeleton elements other than actin. Using double immunofluorescence PLC gamma 1 is shown to be associated with cytokeratin intermediate filaments. The cross-talking of different cytoskeleton elements and their participation in cell signaling is discussed.