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PubMed2003

Solvent dependent and independent motions of CO-horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations.

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.

Arginine↗
PubMed1998

Dehydration-induced conformational changes of poly-L-lysine as influenced by drying rate and carbohydrates.

The conformation of hydrated and air-dried poly-L-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-L-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended beta-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2-3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-L-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (Tg), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest Tg and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.

Desiccation↗
PubMed2002

Determination of the carbonyl groups in native lignin utilizing Fourier transform Raman spectroscopy.

A near-infrared Fourier transform Raman (NIR-FTR) spectroscopic technique was utilized to determine the chemical structure of lignin in a woody matrix. In the NIR-FTR spectra of coniferaldehyde and coniferyl alcohol, the Raman bands for the carbonyl group and the alpha, beta unsaturated bond were detected at 1620 and 1660 cm(-1), respectively. These peaks were also found in the NIR-FTR spectra of chemically synthesized lignins, isolated lignin from conifer wood, and conifer wood meal. Upon the reduction of carbonyl groups in the lignin samples and wood meal, the band at 1620 cm(-1) disappeared; on the other hand, the band at 1660 cm(-1) remained unchanged. However, upon the oxidation of reduced lignin at the benzyl hydroxyl group using dicyanodichrolobenzoquinone, the band at 1620 cm(-1) clearly appeared, strongly suggesting that the band at 1620 cm(-1) can be assigned as a carbonyl marker band. The hydrogenation reaction optimized for the reduction of the unsaturated bond in lignin caused the disappearance of the band at 1660 cm(-1), indicating that the band at 1660 cm(-1) is an alpha, beta unsaturated bond marker band. The change in carbonyl content during the wood decay process was also shown to be monitored using the Raman intensity of the carbonyl marker band. It was indicated that the NIR-FTR spectroscopic techniques were suitable analytical method for a rapid and nondestructive analysis of wood samples.

Cedrus↗
PubMed2005

Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

Alcohols↗
PubMed1997

Identification of a ganglioside recognition domain of tetanus toxin using a novel ganglioside photoaffinity ligand.

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the "1b" substructure (a NeuAcalpha2,8NeuAc group on an internal galactose residue). The domains of tetanus toxin involved in ganglioside binding are known to reside within the carboxyl-terminal half of the toxin's heavy chain ("C fragment"). We developed a novel photoaffinity reagent based upon the structure of the 1b ganglioside GD1b (125I-azido-GD1b) to define the ganglioside-binding domains of tetanus toxin. Using this ligand, we observed radiolabeling of tetanus toxin C fragment which could be specifically inhibited by a ganglioside of the 1b series (GT1b), but not by a non-1b series ganglioside (GM3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with 125I-azido-GD1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by GT1b. Protein sequencing of proteolyzed tetanus toxin C fragment co-migrating with that band revealed the carboxyl-terminal 34 amino acid residues of tetanus toxin. Matrix-assisted laser desorption/ionization mass spectrometry of a photoaffinity labeled synthetic polypeptide representing the 34-amino acid domain revealed modification at a single residue (His1293). We propose that this domain of tetanus toxin is sufficient for ganglioside binding.

Affinity Labels↗
PubMed2004

Formation of molecular complexes by N-methyl-D-aspartate receptor subunit NR2B and ryanodine receptor 2 in neonatal rat myocard.

The N-methyl-d-aspartate (NMDA) receptor is a glutamate gated cation channel prevalent in the postsynaptic membranes of central nervous system neurons. The neurotransmitter receptor complex is thought to represent a tetramer where variable NR2 or NR3 polypeptides form heteromeric assemblies with an obligatory NR1 subunit. Recently, we showed that cardiac myocytes from perinatal rats transiently express the NMDA receptor subunit NR2B, the function of which in heart is unknown. To characterize the cardiac NR2B protein, we determined its subcellular distribution and specific molecular interaction partners. By immunostaining of rat heart tissue slices and acutely dissociated cardiac myocytes, the NR2B antigen was localized at the sarcomeric Z-bands. Using immunoprecipitation of detergent-solubilized NR2B protein and subsequent analysis employing matrix-assisted laser desorption/ionization time of flight mass spectrometry, ryanodine receptor 2 was identified as a molecular interaction partner of the cardiac NR2B polypeptide. Differences in antibody recognition indicate that the cardiac NR2B polypeptide carries a structurally altered C terminus as compared with the NR2B variant prevalent in central nervous system. Based on its localization and protein interaction, the function of cardiac NR2B protein may relate to mechanosensitivity or play a role in the regulation of the contractile apparatus of neonatal heart.

Amino Acid Sequence↗
PubMed2002

Fine structural and immunohistochemical localization of cardiac hormones (ANP) in the right atrium and hypothalamus of the white rat.

Atrial natriuretic peptide (ANP) is a polypeptide hormone secreted primarily by atrial myoendocrine cells. It has diuretic, natriuretic and vasorelaxant effects. ANP has been characterized by non-morphological methods in a number of extra-atrial tissues, particularly the hypothalamus, but little is known of the immunohistochemistry of hypothalamic ANP cells in comparison to atrial ones. Although the presence of ANP-producing cells has previously been confirmed in the right atrium of the rat and other vertebrate species, to our knowledge, this is the first study to demonstrate the presence of these cells in the hypothalamus using a purely morphological method such as electron microscopy. The fine structural and immunohistochemical characteristics of right atrial and hypothalamic ANP positive cells were investigated using immunogold labeling with goat anti-alpha-human ANP (1-28) as primary antibody. Atrial ANP cells were characterized by the presence of membrane-bound electrondense spherical or oval granules with a diameter of about 250 nm. The opaque content of the granules is separated from the limiting membrane by a thin electron translucent band about 20 nm wide. Electron dense crystalloid inclusions were evident within the granule matrix of some atrial ANP granules. Hypothalamic ANP granules were membrane-bound larger in diameter (320 nm), and less electron dense, and lacked crystalloid inclusions. Statistical analyses revealed a significant larger diameter and a significant smaller number of hypothalamic ANP granules compared to atrial ones. The significantly greater number of atrial ANP positive granules suggests a greater volume capacity for the atrial ANP positive granules as compared to the hypothalamic ones. This may indicate that ANP is secreted primarily from the right atrium and to a lesser extent from the hypothalamus; and that both atrial and hypothalamic ANP are closely related in chemical nature and immunohistochemical characteristics. This supports the suggestion that ANP may play the dual role of peripheral hormone and a neurotransmitter or neuromediator.

Animals↗
PubMed1976

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.

Actins↗
PubMed1988

Collagen network remodelling and diastolic stiffness of the rat left ventricle with pressure overload hypertrophy.

This study had two objectives: (a) to determine the accumulation of collagen and its structural remodelling in the hypertrophied rat left ventricle after 4 and 8 weeks of abdominal aorta banding; and (b) to correlate these findings with the diastolic stress-strain relation of the intact myocardium. In comparison to age and sex matched controls, the collagen volume fraction of the hypertrophied myocardium after 4 and 8 weeks of aortic banding increased significantly from 3.5(SD1.0)% to 7.8(4.2)% and 6.2(2.0)% respectively. This accumulation of collagen, or fibrosis, occurred in the absence of myocyte necrosis. Scanning electron microscopy showed increased density and thickness of the collagen weave and tendons. At 4 weeks, light microscopy showed interstitial oedema and disrupted collagen fibrils. Left ventricular diastolic stress-strain relations of both pressure overload groups were significantly steeper than that of the control group. Thus the response of the interstitium to the hypertrophic process that accompanies abdominal aorta banding is a complex process that includes a structural remodelling of the fibrillar collagen matrix and the early appearance of interstitial oedema, each of which may contribute to a rise in the passive stiffness of the intact myocardium.

Animals↗
PubMed1994

Improved procedure for the high-performance liquid chromatographic determination of monohydroxylated PAH metabolites in urine.

An improved high-performance liquid chromatographic (HPLC) method for the determination of 3-hydroxybenzo(a)pyrene (3-OHBaP) in urine was developed. The sensitivity and reproducibility of the technique was greatly improved by the addition of 1 mg/L ascorbic acid to the methanol eluent of the HPLC system. This procedure also eliminated the peak splitting and band broadening of the 3-OHBaP peak otherwise observed. Furthermore, it corrected the urine matrix effect on the slope of standard curves. In fact, in the absence of ascorbic acid in the HPLC system, slopes of standard curves were steeper when prepared in a methanolic extract of control rat urine (121 L.nmol-1) than in methanol only (86 L.nmol-1). Both these slopes were smaller than that obtained with the modified mobile phase (244 L.nmol-1). The effect of the latter on the shape and intensity of the 1-hydroxypyrene (1-OHP) chromatographic peak was also investigated. Again, slopes were greater when the standards, prepared in a methanolic extract of urine, were chromatographed with ascorbic acid (380 L.nmol-1) than without (157 L.nmol-1). Therefore, it seems that ascorbic acid, like certain substances in urine, may act by masking specific adsorption sites--probably uncapped silanol residues on the LC 18 column that can retain free 3-OHBaP and 1-OHP metabolites.

Animals↗
PubMed1989

Serological analysis and characterization of calf thymus ribonuclease H IIb.

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52-kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52-kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52-kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20,000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric. The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N-ethylmaleimide, and exhibits a pH optimum of 9.0-9.5. The enzyme releases oligoribonucleotides with 3'-OH and 5'-phosphate ends, probably in an exonucleolytical manner. The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431-433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.

Animals↗
PubMedDate not supplied

Dexamethasone-induced changes in endometrial growth and inducible nitric oxide synthase during decidualization in rats.

1. The present study investigated the time-dependent inhibitory responses of endometrial growth and inducible nitric oxide synthase (iNOS) to dexamethasone during deciduoma development that was surgically induced on day 4 of pseudopregnancy (PG). 2. Groups of rats (n = 6) were subcutaneously injected with dexamethasone (1.5 mg/rat per day) for 3 days (PG days 1-3, 4-6, 7-9, 10-12 and 12-15). Rats in each group were killed on the last injection day. 3. Dexamethasone produced comparable temporal inhibitory changes in endometrial growth (wet weight, protein and DNA concentrations; P<0.0001) and in iNOS activity (130 kDa protein band), which peaked after PG days 4-6 and 7-9 pretreatments. 4. Endometrial matrix metalloproteinases (72 and 92 kDa) activity profiles displayed maximal reductions (36 and 53%, respectively) following PG days 4-6 pretreatment. Serum progesterone levels were equally (P<0.0001) but asynchronously inhibited by dexamethasone on PG days 9 and 12. 5. Dexamethasone inhibition of endometrial growth and in situ iNOS was most pronounced during decidual development (PG days 4-9). Minor reductions in these endometrial parameters occurred before deciduoma induction (PG days 1-3) and during deciduoma regression (PG days 10-15). 6. These results indicate that, in the endometrium, the iNOS/endogenous nitric oxide system may be linked to the biochemical and metabolic mechanisms responsible for the developmental responsiveness of the deciduoma to dexamethasone exposure. These time-dependent changes in endometrial growth and iNOS apparently were not mediated by progesterone.

Animals↗
PubMed2005

Apparent presence of Ser133-phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross-reactivity of pCREB antibodies with pyruvate dehydrogenase.

Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 alpha-subunit, and E1 beta-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the alpha-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.

Adenosine Triphosphate↗
PubMed2000

Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group".

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the "B. cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.

Bacillus↗
PubMed1987

Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells.

Previous studies established that the chicken ovalbumin gene upstream promoter (COUP) sequence, which lies between -70 and -90 base pairs upstream from the cap site, is essential for the efficient transcription of the ovalbumin gene. A transcription factor which binds to this sequence has been purified from the homologous chicken oviduct cells. The purification scheme starting from oviduct nuclear extract involved a combination of conventional column and sequence-specific DNA affinity chromatography steps. Using gel retardation and DNase I footprinting techniques to assay COUP-binding activity, we achieved extensive purification of this factor. Binding competition studies with the purified factor indicated that it bound specifically to the COUP sequence and that the binding could be competed for only by the promoter DNA fragments or synthetic oligonucleotides containing the COUP sequence. The purified protein preparation showed multiple polypeptide bands on polyacrylamide gel electrophoresis. Renaturation of separated polypeptides after extraction from the gel matrix was carried out. The majority of renatured polypeptides exhibited specific binding to the COUP sequence.

Animals↗
PubMed1994

Tenascin is synthesized and secreted by rat mesangial cells in culture and is present in extracellular matrix in human glomerular diseases.

Tenascin (TN) is a large oligomeric protein recently described as a component of the extracellular matrix. The distribution of TN in adult kidney tissue has not been adequately evaluated, but preliminary data have suggested that TN is variably seen in rare mesangial areas and in stroma surrounding some tubules. The enlargement of the mesangial matrix (mesangial sclerosis) is a common feature of many renal diseases and is thought to be partially related to oversynthesis of the normal components of the mesangial matrix (collagen type IV, laminin, fibronectin, and heparan sulfate proteoglycans) by mesangial cells. However, the possibility that mesangial cells are also the source of other extracellular matrix proteins that participate in the process of mesangial sclerosis has not been explored. In this study, the synthesis of TN by cultured rat mesangial cells was documented by the following observations: (1) Northern hybridization of total RNA extracted from mesangial cells showed two distinct species of TN mRNA; (2) immunoblotting of the protein extracted from the conditioned medium demonstrated four TN protein bands; (3) immunoblotting of the protein extracted from the mesangial cell lysate demonstrated at least four TN protein bands; and (4) immunohistochemical techniques identified TN within the cytoplasm of mesangial cells and in the surrounding extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

[Genetic diversity of yam (Dioscorea opposita Thunb) detected by ISSR markers].

Genetic diversity of 28 cultivars of yam (Dioscorea opposita Thunb) was assessed by means of Inter-simple sequence repeat (ISSR) markers. The results showed that seven proper primers, with rich polymorphism, could be selected from a total of forty four ISSR ones; distinct differences appeared among 28 cultivars amplified bands, and the rate of polymorphic bands was 83.01%; Shannon's Information index was 0.3191; a Jaccard's genetic similarity matrix and a dendrogram for these cultivars were formed, in which they could be divided into four groups: Group 1 was composed of D. opposita. cv. Ribenbai, D. opposita. cv. Huashanyao and D. opposita. cv. Ribenyuan; Group2 contained D. opposita. cv. Xiaoye; Group 3 contained D. opposita. cv. No.1 Songye; other 23 cultivars were put into Group4. PCA(Principal component analysis) was employed to evaluate the resolving power of the markers to differentiate among them. This laid the foundation of the identification of yam cultivars and the efficient use of its germplasm resources.

DNA, Plant↗