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PubMed1987

Rebinding and relaxation in the myoglobin pocket.

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.

Carbon Monoxide↗
PubMed2001

Clonal variation in phenotype and life span of human embryonic fibroblasts (MRC-5) transduced with the catalytic component of telomerase (hTERT).

Expression of telomerase (hTERT) in certain cell types has been shown to extend cellular life span without malignant transformation. We studied the phenotype of 26 telomerase-transduced fibroblast clones (TTFC) generated from a mass culture of hTERT retrovirally transduced MRC-5 cells. About two-thirds of the transduced clones senesced at the expected time or shortly thereafter, despite high levels of expression of telomerase and telomere length maintenance. The remaining one-third of the clones were "immortalized" (followed for over 200 cumulative population doublings). All clones maintained a nontransformed phenotype: contact inhibition, anchorage dependency, lack of tumor formation in nude mice, dose dependency to serum and growth factors, low expression of a matrix metalloproteinase associated with metastatic invasion (MMP-9) and high expression of its inhibitor TIMP-1, and no cytogenetic abnormalities by G-banding. In addition, fibroblast-specific biological parameters, such as colony size, production of collagenase, and response to MMC and gamma radiation were tightly regulated at the clonal and subclonal levels.

Animals↗
PubMed1997

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath.

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

Actin Cytoskeleton↗
PubMed2005

Technical note: the design of a stereotactic frame for direct MRI-anatomical correlation of the brachial plexus.

The purpose of this study was to identify optimal magnetic resonance imaging (MRI) conditions to visualize discrete alterations of brachial plexus components, as part of a biomechanical study of minor nerve compression syndromes. A method was developed allowing direct comparison between the MRI image and the subsequently obtained matching anatomic section of the same specimen. We designed a stereotactic frame to obtain the precise orientation of the MRI plane with reference to the specimen and adapted a vertical band saw for multiplanar sectioning of cadaveric specimens. Two cadaveric upper quadrants were examined by MRI (TR 450 ms, TE 13 ms, pixel matrix 512 x 512 and FOV 23-26 cm) and anatomical slices were produced. One specimen was sectioned axially, while the second specimen was sectioned in an oblique plane corresponding to the natural longitudinal axis of the upper part of the brachial plexus. MR images and the corresponding slices exhibited a strong correlation. This correlation was checked by using vitamin A pearls as landmarks. MR images revealed more detail after the correlating anatomical slices were analyzed. The present study shows that the method is suited for direct MRI-anatomic comparison of the brachial plexus and is also proposed for application to other topographical regions.

Acromioclavicular Joint↗
PubMed1980

A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase.

We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.

Acetylation↗
PubMed1991

Autoradiographic, electrophoretic, and immunocytochemical studies of glycoproteins of the rabbit iris.

L-[3H]fucose was injected either intravitreally or intra-aqueously into adult rabbits which were killed at several time points after injection. SDS-polyacrylamide gel electrophoresis and fluorography of iris extracts revealed that most of the proteins are glycoproteins containing fucose residues. Autoradiography of semi-thin histologic sections demonstrated that glycoprotein synthesis was most prominent in the epithelium of the iris, while little protein synthesis was evident in the stroma of the iris. The results of these experiments indicated that the glycoproteins of the iris undergo renewal. The protein band pattern of the iris extracts was very similar to that of extracts of the ciliary body. The high-molecular-weight cartilage matrix glycoprotein (CMGP), an intrinsic component of the ciliary body, vitreous, and aqueous humor, was detected by immunohistologic studies only in the stroma of the iris. The results of immunohistochemical analyses of the eyes of young rabbits (1-21 days old), in addition to the autoradiographic findings, strongly suggest that CMGP is not an intrinsic glycoprotein of the iris stroma, at least in this species.

Animals↗
PubMed1994

Energy migration in allophycocyanin-B trimer with a linker polypeptide: analysis by the principal multi-component spectral estimation (PMSE) method.

Energy migration processes in allophycocyanin-B trimer with a linker polypeptide were analyzed using the principal multi-component spectral estimation (PMSE) method, which does not require assumption of component number, decay function, or the spectral band shape. We determined the number of spectral components showing independent kinetic behavior by the eigen-value of an auto-correlation matrix, and further the spectra of the components and their rise and decay curves. Two decay components were resolved at 20 degrees C: one corresponded to the decay of one type of beta-84 chromophore, and the other to the decay from the thermally equilibrated state between another type of beta-84 chromophore and the alpha-allophycocyanin B chromophore. An additional slow decay process was resolved at -196 degrees C. We also compared the component spectra obtained using the PMSE method with the decay-associated spectra obtained using the global analysis.

Cyanobacteria↗
PubMed1991

A RNA virus in cells from Culicoides variipennis.

A virus was detected in cells (designated CuVa) cultured from one laboratory colony of the biting midge, Culicoides variipennis. By electron microscopy (30 nm), nonenveloped, icosahedral virions arranged separately and in crystalline matrix arrays were seen in the cytoplasm but not in the nucleus of CuVa cells. Separation by 10% polyacrylamide gel electrophoresis revealed multiple bands of viral-induced double-stranded RNA. Inoculation of this virus onto different cell lines and intracranially into suckling mice revealed no detectable pathology. Immunoperoxidase staining using polyclonal antibody determined that the virus is infectious to toad cells, bovine endothelial cells, bovine kidney cells, mosquito cells, and cells (designated KC) initiated from another laboratory colony of C. variipennis. KC cells infected with this virus were coinfected with bluetongue virus with no decrease in bluetongue virus titer.

Animals↗
PubMed1993

Estimation of the secondary structure and conformation of bovine lens crystallins by infrared spectroscopy: quantitative analysis and resolution by Fourier self-deconvolution and curve fit.

The secondary structure of six bovine lens protein fractions (two alpha, three beta and one gamma-crystallin) are examined in solution and in solid forms for the first time using FTIR spectroscopy. Films of the nuclear and cortical regions of the bovine lens are also examined. The structure is quantitatively estimated from the vibrational analysis of the resolution-enhanced amide-I profile achieved by Fourier self-deconvolution and linear least-squares curve-fit algorithm. All the protein fractions fold predominantly in a beta-pleated sheet structure with little or no alpha-helical domains in solution or in lyophilized solid form. These proteins also retain their predominant beta-sheet conformation in the cellular phospholipid environment of the lens, in conformity with the structure obtained for all the mammalian species examined to date. Despite structural homology, vibrational data indicate subtle structural differences within each class of the crystallins probably due to presence of several minor substructures/subconformations. Substantial high amounts of turns (approx. 40%) observed in the beta-fractions may have a fundamental implication in stabilizing the tertiary structure of the uniquely folded-proteins vital for the transparency of the lens. These proteins in solid KBr-matrix undergo a major structural change, induced primarily by ionic interactions which refold them in a helical conformation. IR spectroscopy together with band-narrowing procedures has proven to be an effective tool to obtain structural information of proteins in solution, as solid substrates or in a complex biological tissue, such as ocular lens.

Algorithms↗
PubMed1994

Calculation of the circular dichroism spectrum of cyclo-(L-tyr-L-tyr) based on a molecular dynamics simulation.

Theoretical calculations of CD spectra have generally assumed a single conformation, or a small number of conformers with Boltzmann averaging. Solvent effects on both the conformation and the CD have been neglected. In this work, we have calculated the CD spectrum of cyclo(L-Tyr-L-Tyr) in aqueous solution, taking dynamics and solvation into account. Starting geometries with chi 1 approximately 300 degrees or 60 degrees for both Tyr side chains were derived from MNDO/MOPAC, followed by energy minimization using GROMOS. After addition of 368 water molecules, the system was simulated for 1000 ps at 300 K using GROMOS. In addition to the starting conformer, two other conformers were observed during each simulation. However, each trajectory gave a distinct set of conformers. Rotational strengths were calculated for the cyclic dipeptide at each ps along the trajectories, using the matrix method. The CD spectra calculated from these rotational strengths were averaged over the trajectories. Agreement is very good for the strong negative band near 200 nm, while for the lower energy bands (near 230 and 280 nm), the signs are correct, but the magnitudes are too low. The spectrum calculated from a Boltzmann-weighted average over the in vacuo MNDO/MOPAC conformers was in poor agreement with experiment. Although the solvent did not significantly affect the rotational strength calculated for a given conformer, it is essential to include the solvent in the MD simulations because it affects the relative energies of the conformers and promotes transitions among them.

Chemical Phenomena↗
PubMed2006

Inhibition of prolyl 4-hydroxylase prevents left ventricular remodelling in rats with thoracic aortic banding.

BACKGROUND: Pressure overload leads to myocardial remodelling with collagen accumulation, left ventricular hypertrophy (LVH), neurohormonal activation and myocardial dysfunction. Prolyl 4-hydroxylases (P4H) are involved in collagen maturation. Inhibition of P4H has been shown to prevent LV remodelling and improve survival post-myocardial infarction. AIM: To evaluate the role of P4H in pressure overload-induced myocardial remodelling. METHODS: Male Wistar rats underwent thoracic aortic banding (AoB) and were treated with a P4H inhibitor (P4HI) or vehicle (control). Echocardiography and haemodynamic measurements were performed after 4 weeks. Collagens, matrix metalloproteinases (MMP), tissue inhibitors of MMPs (TIMP), growth factors and neurohormonal markers were quantitated in LV samples. RESULTS: AoB led to LVH, increased LV enddiastolic pressure (LVEDP) and decreased contractility compared to sham. P4HI reversed these effects. AoB increased collagen I and III expression, which was normalized by P4HI. AoB led to deregulation of matrix remodelling enzymes, enhanced expression of growth factors and activation of the endothelin system. P4HI partially prevented deregulation of the MMP/TIMP system, inhibited upregulation of growth factors and normalized AoB-induced ECE-1 and ETB expression. CONCLUSIONS: P4HI leads to an improvement of AoB-associated LV dysfunction and reduces imbalance of extracellular matrix turnover and hypertrophy-associated gene expression. P4H inhibition could therefore be of value in treatment of myocardial remodelling accompanying pressure overload hypertrophy.

Animals↗
PubMed2006

Melanotic macule of nail unit and its clinicopathologic spectrum.

The clinical and histologic spectrum of melanotic macule of the nail unit is examined and the differences in the clinical appearance of longitudinal melanochychia caused by melanotic macule and by other kinds of proliferations of melanocytes are assessed. We observed that the clinical appearance of the pigmented band was of little help in establishing the underlying basic pathologic process. This underscores the importance of obtaining a biopsy of the nail matrix in patients who present with solitary longitudinal melanonychia.

Adolescent↗
PubMed2004

Striosomes are enriched in glutamic acid decarboxylase in primates.

The compartmental distribution of glutamic acid decarboxylase (GAD) in the striatum was investigated in squirrel monkeys and rats with antibodies raised against the two isoforms of this enzyme (GAD65 and GAD67) and with calbindin D-28k (CB) and/or micro-opiate receptor (MOR) as striosomal markers. In primates, immunostaining for both GAD65 and GAD67 was much more intense in striosomes than in the surrounding matrix. A thin immunoreactive strip of GAD labeling was also present in the dorsolateral part of both caudate nucleus and putamen. This narrow band appears to correspond to the so-called subcallosal streak (SS) found in rodent striatum. Although the immunostaining intensity for the two enzymes was similar at pallidal level, that for GAD65 was more intense than that for GAD67 at the striatal level. The GAD immunostaining was more uniformly distributed in the rat striatum, which did not display GAD-rich patches that corresponded to MOR-positive striosomes. Moreover, in contrast to the findings obtained in monkeys, the subcallosal streak in rats was less intensely stained for GAD than for the remaining regions of the striatum. These results reveal that GAD65 and GAD67 are faithful markers of striosomes in primates but not in rodents. They suggest the existence of a significant species difference between rodents and primates in respect to the chemical organization of the striatum, a difference that should be taken into account when using rodents as animal models to study the functional organization of the basal ganglia and the pathogenesis of neurodegenerative diseases that affect the striatum.

Animals↗
PubMed2005

Detection of signal synchronizations in resting-state fMRI datasets.

In this paper, we propose a generic framework for the analysis of steady-state fMRI datasets, applied here to resting-state datasets. Our approach avoids the introduction of user-defined seed regions for the study of spontaneous activity. Unlike existing techniques, it yields a sparse representation of resting-state activity networks which can be characterized and investigated fairly easily in a semi-interactive fashion. We proceed in several steps, based on the idea that spectral coherence of the fMRI time courses in the low frequency band carries the information of interest. In particular, we address the question of building adapted representations of the data from the spectral coherence matrix. We analyze nine datasets taken from three subjects and show resting-state networks validated by EEG-fMRI simultaneous acquisition literature, with low intra-subject variability; we also discuss the merits of different (rapid/slow) fMRI acquisition schemes.

Algorithms↗
PubMed1998

The two spectroscopically different short wavelength protochlorophyllide forms in pea epicotyls are both monomeric.

The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.

Journal Article↗
PubMed1986

Purification of larvicidal protein from Bacillus sphaericus 1593.

Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis. Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae. IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix. The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column. The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions. This protein contained 12% carbohydrates. The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus.

Bacillus↗
PubMed1999

Metal-matrix interface in reinforced glass ionomers.

OBJECTIVE: In reinforced materials, interfacial bonding is critical for efficient transfer of stress from the matrix to the reinforcement. The goal of this study was to characterize the physical and chemical nature of this interface in three metal-reinforced glass ionomers. Two of them were commercial, ESPE-Ketac Silver (KS) and GC-Miracle Mix (MM), and the third, EX, was experimental. METHODS: The techniques of scanning electron microscopy (SEM) and infrared spectroscopy (IR) were used to accomplish the stated goal. SEM analysis utilized polished sections of set cylindrical specimens of each material prepared from their respective powder and liquid components. The glass and the liquid in EX were the same as in MM. The reinforcing agent in EX, 50% by weight, was an Ag-base spherical alloy similar in composition to that in MM. For EX, the alloy was oxidized to promote its bonding to the matrix. The specimens for IR study were prepared as follows. The metallic powders of each material were mixed with the corresponding liquid in excess, stored at 37 degrees C for 1 h, washed with warm water (60 degrees C), filtered and dried. The untreated metallic powders served as controls for IR analysis. RESULTS: SEM revealed a distinct halo shaped internal reaction layer surrounding each alloy particle in EX. A similar layer was not seen in MM and KS. The alloy-matrix interface was continuous and gap-free in EX. In contrast, gaps separating matrix from respective reinforcements were conspicuous features in MM and KS. The IR spectrum of the liquid treated EX alloy powder showed absorbency bands characteristic of unreacted carboxyl groups and carboxylate salts. These bands were absent in the IR spectra of all other powders--treated and untreated. SIGNIFICANCE: The absence of interfacial bonding in MM and KS demonstrated in this study provides a reason why these two materials, in spite of metal addition, have not proved to be any stronger or more durable than their metal-free counterparts. A means of creating interfacial bonding presented here could be useful in the design of improved reinforced glass ionomer materials.

Aluminum↗
PubMed2002

Modifications on PVC films by ultrasound.

PVC films submitted to ultrasonic irradiation presented structural changes as probed by infrared measurements. These measurements showed some infrared bands alterations attributed to the increase concentration of some conformational isomers of PVC and the presence of some new species in the macromolecular matrix.

Journal Article↗