Inter-hemispheric scissure, a rare location for a traumatic subdural hematoma, case report and review of the literature

The pBR322 recombinant plasmids DNA products, which purified by phenol-extract and equilibrium centrifugation in CsCl-ethidium bromide gradients, have been shown as mainly spatial conformation of covalently closed circular DNA (i.e. cccDNA) by agarose gel electrophoresis for homogeneity detection. Laser Raman spectra in the region 450-1750 cm-1 have been obtained for the circular double-stranded plasmids DNA molecule in an aqueous solution, indicating not only contain those marker peaks of secondary structure as in conventional B-form of DNA, but also present both 854 and 1083 cm-1 diagnostic bands of reflecting the vibration state of deoxyribosyl phosphodiester backbone. The present analysis have demonstrated relationship between the superhelical state for cccDNA and the two conformational marker bands that can be considered as the tertiary structure marker on plasmids DNA. Because of markedly Raman hypochromicity of charecter band 1378cm-1 of dT in contrast to liear DNA molecule, as well as the carbonyl double bond vibration line of dT have shifted to higher wave number position, the base stacking analysis represent occurrence of both increase in staking reaction activity of dT, and injury of a number of the Hoogsteen hydrogen bond between dA and dT with exist supercoil structure in cccDNA.     

Characterization of vinyl-substituted, carbon-carbon double bonds by GC/FT-IR analysis

Vapor-phase infrared spectra allow the determination of the stereochemistry of carbon-carbon double bonds conjugated with a vinyl group. Cis and trans isomers of unsubstituted 1,3-alkadienes can be differentiated on the basis of the differences observed in the 900-1000 cm-1 region (spectra of cis isomers show two bands at 993 and 906 cm-1, while those of trans compounds show three absorptions at 998, 949, and 902 cm-1) and the 1590-1650 cm-1 region (the C=C stretch bands are observed at 1595 and 1642 cm-1 for cis compounds and at 1604 and 1650 cm-1 for trans compounds). Compounds bearing CH2=CHC(CH3)=CHCH2- and CH2=CHC(=CH2)-CH2- structural moieties, referred to as alpha- and beta-type compounds, are frequently encountered as natural products. For compounds bearing alpha-type groups, the cis/trans configuration of the trisubstituted double bond can be determined unambiguously. An absorption at 3095-3091 cm-1, for the =CH2 stretch vibration, is common to both of these groups; however, due to the presence of two =CH2 groups, the relative intensity of the band is much higher for beta-type compounds. For alpha-type compounds, a cis configuration at the C-3 carbon atom is characterized by a =CH2 wag absorption at 907-906 cm-1. For beta-type compounds and 3E-alpha-type compounds, this band appears at 899-897 cm-1. In addition, a wavy "fingerprint" pattern with two minima at 1632 (low intensity) and 1595-1594 cm-1 (high intensity) is characteristic for beta-type compounds. Our generalizations are based on spectra of cis and trans ocimene, myrcene, and dehydration products of many 3-methyl-1-alken-3-ols. Six isomers of farnesene can be characterized by GC/FT-IR. Furthermore, gas-phase IR allows the determination of the configuration of the trisubstituted double bond at C-3 in alpha-type farnesene congeners. For example, the homo- and bishomofarnesene isomers from Myrmica ants were shown to include a 3Z bond.     

FT infrared and Raman investigation of saccharide-phosphatidylcholine interactions using novel structure probes

Conformational consequences of adduct formation between saccharides (trehalose, glucose, raffinose) and sorbitol with dipalmitoylphosphatidylcholine (DPPC) in multibilayers are revealed by relative intensity changes of the band components corresponding to the nu asN(CH3)3 and nu sC-N(CH3)3 stretching modes of the choline chain terminal and those of the nu C = O band. The conformational sensitivity of those modes was demonstrated previously (J. Grdadolnik et al., Chem. Phys. Lipids 65 (1993) 121) and used to demonstrate the effects of stepwise hydration of phosphatidylcholines. The latter are compared with the effects of saccharide binding and found to be qualitatively similar, but not identical. The same is true of the low frequency shifts of the nu asPO2- vibration: the shifts due to saccharide binding correspond to the binding of six to seven water molecules per phosphate which is about 20 cm-1 less than the shift caused by full hydration. A particularly interesting finding concerns the appearance of two bands in the nu asPO2- region of the DPPC-saccharide adducts. The relative intensities of the two bands (1243 and 1223 cm-1) change on additional hydration; it is the one at 1223 cm-1 that prevails at high hydration levels. Major changes in saccharide conformation are not detectable but minor differences between the DPPC bound and crystal spectra are observed.     

A relative energy failure is associated with low-Mg2+ but not with 4-aminopyridine induced seizure-like events in entorhinal cortex

Interactions of the primary quinone acceptor QA of photosystem II (PS II) with surrounding amino acid residues were studied by analysis of FTIR difference spectra of QA upon its photoreduction (QA-/QA). Structural coupling with a His side chain was revealed by identifying the imidazole bands in the QA-/QA spectrum using the PS II core complexes from Synechocystis PCC 6803 in which both of the two imodazole nitrogens of His side chains were specifically labeled with 15N. Strong hydrogen bonding of the imidazole NH was shown by (i) the presence of several peaks at 2600-3000 cm-1, which arise from Fermi resonance of harmonics or combinations of imidazole ring modes with the hydrogen bonding NH stretching vibration, and (ii) the 1179 cm-1 band, which can be assigned to the mode including NH deformation, is at a frequency significantly higher than the corresponding 1151 cm-1 band of model compounds 4- and 5-methylimidazole in aqueous solution. Also, the presence of the bands specific to the Npi-protonated state at 1109/1102/1090 and 1359 cm-1 suggests that the QA-coupled His is protonated at the Npi site. These results are in good agreement with the model of QA interaction in which His215 (D2), which coordinates to the non-heme iron at Ntau, is hydrogen bonded to the QA carbonyl through the Npi-H bond. In contrast, no bands of Trp side chains were detected in the QA-/QA spectrum upon labeling of the indole ring of Trp residues with indole-d5. This result indicates that Trp254 (D2), which corresponds to Trp252 (M) of the bacterial reaction center that is located in van der Waals contact with QA, is not strongly coupled with QA in PS II. Probably, the predicted pi-pi interaction is not strong enough to influence the vibrations of the indole ring of Trp upon QA reduction, or Trp254 (D2) is located rather far from QA in PS II.     

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

Strain diversity in the transmissible spongiform encephalopathies (TSEs) has been proposed to be determined by variations in the conformation of the abnormal, protease-resistant form of prion protein (PrP-res). We have investigated whether infection of hamsters with three TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters infected with the hyper (HY), drowsy (DY), and 263K TSE strains yielded similar SDS-polyacrylamide gel electrophoresis profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a fragmentation pattern that was distinct from that of the other two strains. Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626 and 1636 cm-1 both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 cm-1 and stronger band intensity at both 1616 and 1694-1695 cm-1. Because absorbances from 1616 to 1636 cm-1 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases.     

Laser Raman investigation of the conformation of human immunoglobulin G

Laser Raman spectra of human immunoglobulin G in neutral solution, as well as in the lyophilized and alkaline-denatured states are presented. In the spectrum of the native protein, the amide III band appears at 1240 cm-1 and is assigned to the presence of beta-sheet structure. From its intensity, using a procedure described in this paper, we evaluate the beta-structure content to 37 +/- 4%. This result is supported by the strong amide I' band at 1667 cm-1 and by the presence in the spectra of two bands at 991 and 1078 cm-1, respectively assigned to the C-C and C-N skeletal stretching modes. The differences between the spectrum of the lyophilized powder and that of the solution show that the lyophilization process induces conformational changes that perturb the local environment of some of the tryptophan residues and alter the secondary structure of immunoglobulin G. The beta-structure appears to be more uniform and more abundant in solution. When the protein is denatured at pH 11, the amide III and amide I'bands, which become weaker and broader, shift in frequency from 1240 to 1248 cm-1 and from 1667 to 1656 cm-1 respectively. These changes indicate a decrease in the amount of beta-structure and a transition toward a much more disordered conformation. During the denaturation, the intensities of many bands of the aromatic chromophores change, notably the tryptophan peaks at 879, 1359 and 1573 cm-1.     

Immunoassays based on surface-enhanced infrared absorption spectroscopy

A new type of biosensor for pathogens has been developed. The sensor produces spectral fingerprints of biological systems by using surface-enhanced infrared absorption (SEIRA) spectroscopy. Antibodies were immobilized onto a 10-nm-thick film of gold which had been previously deposited on a Si wafer. SEIRA spectra of the antibodies measured in the external reflection mode exhibited two new bands at 1085 and 990 cm-1. These new bands were observed with p-polarized radiation but were absent with s-polarized radiation. The spectrum of water on the surface of the sensor was observed under both directions of polarization. The sensor was first tested with a model system consisting of glucose oxidase (GOX) and the antibodies for glucose oxidase (anti-GOX). In addition to the bands due to the anti-GOX at 1085 and 990 cm-1, new bands were observed at 1397, 1275, and 930 cm-1 when the GOX antigens were present. The same type of sensor was prepared for Salmonella (SAL) by immobilizing antibodies for Salmonella (anti-SAL) on a gold-surfaced Si water. The SEIRA spectra for anti-SAL antibodies were very similar to those for anti-GOX, with bands at 1085 and 990 cm-1; however, a sharp new band was observed at 1045 cm-1 after the sensor was exposed to the SAL antigens. In addition to specific new bands due to antigens, both GOX and SAL sensors exhibited changes in the regions of water absorptions at approximately 3500 and 850 cm-1 when the antigens were present.     

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.     

An FT-Raman spectroscopic investigation of dentin and collagen surfaces modified by 2-hydroxyethylmethacrylate

Although 2-hydroxyethylmethacrylate (HEMA) is commonly used for adhesive bonding to dentin, its role in promoting adhesion is not completely understood. Here, we use FT-Raman spectroscopy to elucidate further the nature of the interaction of HEMA with dentin. Ground dentin was exposed to 2.5% (w/w) nitric acid, washed, dried in air, and treated with HEMA. The samples were then sequentially washed with distilled water, with FT-Raman spectra being obtained after different wash times. Hydroxyapatite and bovine type I collagen were similarly treated with HEMA except for the acid exposure. The FT-Raman spectra of these samples were also recorded. The spectra of HEMA-treated water-washed dentin and collagen revealed the following changes: (1) The band intensities of HEMA absorbed on dentin and collagen decreased with increasing wash times (2) the nu(C=O) and nu(CCO) modes of HEMA at 1718 and 607 cm-1, respectively, either disappeared or decreased after extensive washing; (3) the nu (C=C) (1640 cm-1) and delta (=CH2), (1403 cm-1) bands exhibited minor variations in band position and relative intensity. These results demonstrate that HEMA interacts with dentin both physically and chemically. The chemical interaction can be interpreted by either hydrogen bonding or the formation of a new bond to the ester group of HEMA.     

Stability of triple-helical poly(dT)-poly(dA)-poly(dT) DNA with counterions

Structural conformation of triple-helical poly(dT)-poly(dA)-poly(dT) has been a very controversial issue recently. Earlier investigations, based on fiber diffraction data and molecular modeling, indicated an A-form conformation with C'3-endo sugar pucker. On the other hand, Raman, solution infrared spectral, and NMR studies show a B-form structure with C'2-endo sugars. In accordance with these experimental results, a theoretical model with B-form, C'2-endo sugars was proposed in 1993. In the present work we investigate the dynamics and stability of the two conformations within the effective local field approach applied to the normal mode calculations for the system. The presence of counterions was explicitly taken into account. Stable equilibrium positions for the counterions were calculated by analyzing the normal mode dynamics and free energy of the system. The breathing modes of the triple helix are shifted to higher frequencies over those of the double helix by 4-16 cm-1. The characteristic marker band for the B conformation at 835 cm-1 is split up into two marker bands at 830 and 835 cm-1. A detailed comparison of the normal modes and the free energies indicates that the B-form structure, with C'2-endo sugar pucker, is more stable than the A-form structure. The normal modes and the corresponding dipole moments are found to be in close agreement with recent spectroscopic findings.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Effect of ethanol on the protein secondary structure of the human gastric mucosa, in vitro

The effect of ethanol on the secondary conformational structure of proteins of the human gastric mucosa was investigated by attenuated total reflection/Fourier transform infrared (ATR/FT-IR) spectroscopy. The IR peak intensity and position of each structural component of gastric mucosa was found to change significantly with the ethanol concentration and length of exposure. The peak intensity due to the beta-sheet and/or beta-turn conformational structure in amide I and II bands of gastric mucosa clearly increased after treatment with ethanol. Moreover, the peak at 1635 cm-1 shifted to 1630 cm-1 after treatment with 40% ethanol for 3 h, or 80% ethanol for 1 h, and a distinct shoulder also appeared at 1643 cm-1. This shift occurred more rapidly and was more pronounced after exposure of mucosa to 80% ethanol, compared with the effect of 40% ethanol, but the alpha-helical structure at the amide I and II bands was not influenced by either concentration of ethanol. Ethanol treatment might also transform the secondary structure of amide III in gastric mucosa from an alpha-helix to a mainly random coil with extensive unfolding. The absorption between 1180 and 980 cm-1, which is assigned to glycoprotein structure, was also reduced after treatment with ethanol. This strongly indicates that ethanol influences the conformation of the lipids and proteins of human gastric mucosa, leading to their deformation.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Fourier transform infrared spectroscopy characterization of the lamellar and nonlamellar structures of free lipid A and Re lipopolysaccharides from Salmonella minnesota and Escherichia coli

The structural polymorphism of free lipid A and deep rough mutant lipopolysaccharide (LPS Re) from Salmonella minnesota strain R595 and Escherichia coli strain F515 was characterized by Fourier transform infrared (IR) spectroscopy. For this, the beta <--> alpha phase states and the three-dimensional supramolecular structures, the latter deduced from small-angle synchrotron radiation x-ray diffraction, were investigated at different water contents, Mg2+ concentrations, and temperatures. The analysis of the IR data for vibrations originating from the hydrophobic moiety shows that the beta <--> alpha acyl chain melting is strongly expressed only for the stretching and scissoring modes of the methylene groups. Vibrational groups originating from the interface region sense the acyl chain melting well (ester carbonyl bands) or only weakly (amide bands), and those resulting from the pure polar moiety not at all. From the x-ray data, the existence of lamellar (L), different cubic, and, for lipid A and LPS R595, also inverted hexagonal (HII) structures could be proven in the temperature range 20-80 degrees C with cubic <--> cubic and cubic <--> HII transitions for the Mg(2+)-free and L <--> HII transitions for the Mg(2+)-containing samples. These structural transitions can be characterized most readily by specific changes of the vibrational bands resulting from the interface region: the ester carbonyl and the amide bands. The magnitude of the changes corresponds to that of the structural rearrangement, i.e., is highest for the L <--> HII, lower for the cubic <--> HII, and lowest for the cubic <--> cubic transitions. The structural transitions are only marginally expressed for vibrational bands of the hydrophobic moiety. Similarly, the band contours of vibrations from the hydrophilic region are no indicators of the structural reorientations except for the carboxylate bands of LPS Re. Particularly the stretching vibrations of the phosphate groups are nearly completely invariant; the absolute values of their half bandwidths, however, differ significantly for lipid A and LPS Re, which seems to be of biological relevance. The ability of IR spectroscopy to detect supramolecular changes also beyond the measurability by x-ray diffraction, i.e., at water contents > 95 to 99.5%, is demonstrated.     

Structure and architecture of the bacterial virus fd. An infrared linear dichroism study

Oriented gels of intact bacterial virus fd have been invetigated by infrared linear dichroism. Infrared absorption band maxima and dichroism indicate an alpha-helix content of the major coat protein of 95-100%. The alpha-helical rods of the coat protein are aligned parallel to the long axis of the virion with an inclination roughly estimated to approximately 37 degree. The presence of DNA infrared bands at 968, 885, 830 and 799 cm-1, the absence of a band at 860 cm-1 and the perpendicular polarization of the symmetric PO-2 stretching vibration at 1085 cm-1 are all indicative of a B-type backbone conformation in the single-stranded DNA. We find no evidence for specific interaction between aromatic side groups (phenylalanine, tyrosine) and the DNA bases. Our results independently confirm most features of the model of Marvin and co-workers [2,15 ] based on low-resolution X-ray diffraction studies. However, our findings contradict their suggestion of an A-type DNA in the bacterial virus fd. Two results are consistent with rigid and stable order in the virus. First, over a 4-day period, 65% of the peptide hydrogens remain unexchanged with deuterium. Second, changes in the relative humidity of the sample do not result in any shifts in the DNA spectrum that are characteristic of free DNA.     

Conformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopy

The 13C labeled lipid 1[1'-13C]DPPS-NH4+ and its metal salts were used to unambiguously assign all carbonyl vibrations in the infrared spectrum of phosphatidylserines. It is shown that the C=O stretching band at 1741 cm-1 of phosphatidylserines previously assigned to the sn-1 C = O vibration contains contributions from both the sn-1 and the sn-2 carbonyls. The C=O stretching band at frequencies between 1715 and 1730 cm-1 previously assigned to the sn-2 C=O vibration also contains contributions from both carbonyl groups. The frequency dependence observed with the ester carbonyls primarily reflects hydrogen bonding and the polarity of the immediate vicinity. Conformational changes are accounted for in terms of frequency shifts if the conformational change involves the disposition of the C=O groups and in turn the hydrogen bonding properties. The infrared spectra of phospholipids dispersed in aqueous medium in the liquid crystalline state are inconsistent with a simple phospholipid conformation, e.g., with a conformation as found in the single-crystal structure of 1,2-dimyristoyl-sn-phosphatidylcholine and 1,2-dilauroyl-rac-phosphatidylethanolamine. The spectra support the hypothesis proposed earlier (Hauser et al., Biochemistry, 1988) on the basis of existing single-crystal phospholipid structures and NMR evidence. The hypothesis states that several conformations are present in liquid crystalline phospholipid dispersions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells

PURPOSE: To identify matrix metalloproteinases (MMPs) released by ciliary smooth muscle cells in vitro and to determine whether MMP release is altered by exposure to prostaglandins (PGs). METHODS: Human ciliary smooth muscle cells were grown to confluence in monolayer cultures and treated with PGF2 alpha, 11-deoxy-PGE1, or PhXA85 (the nonesterified analogue of PhXA41) for 12 to 72 hours. The activity of MMP in the medium was assayed using gelatin and casein zymography. Identification of the specific MMP associated with each band was made by Western blot analysis. Band intensity, which reflects activity, was measured with a scanning laser densitometer. RESULTS: Three major bands appeared in the gelatin zymographs at positions corresponding to molecular weights of 62 kDa, 68 kDa, and 97 kDa. A single band at 50 kDa predominated in the casein zymograms. Substitution of EDTA for calcium and zinc in the development solution eliminated the appearance of these bands, indicating that they reflect MMP activity. Immunoblotting, using MMP-specific antibodies, confirmed that the three bands in the gelatin zymographs were MMP-1, MMP-2, and MMP-9, respectively; the single band in the casein zymographs was MMP-3. Treatment with 200 nM PGF2 alpha, 11-deoxy-PGE1, or PhXA85 for 72 hours increased the combined density scores for MMP-1 and MMP-2 by 37%, 64%, and 27%; the density scores for MMP-9 by 268%, 253%, and 125%; and the density scores for MMP-3 by 35%, 71%, and 22%, respectively. CONCLUSIONS: These results indicate that ciliary smooth muscle cells can secrete MMP-1, MMP-2, MMP-3, and MMP-9. In addition, exposure to PGF2 alpha, 11-deoxy-PGE1, or PhXA85 increases production of all four MMPs. These observations support the hypothesis that increased MMP production by ciliary muscle cells has a role in increasing uveoscleral outflow facility after topical PG administration.     

Infrared Fundamental and First Hot Bands of O12C17O Isotopic Variants of Carbon Dioxide

Infrared spectra of 16O12C17O, 17O12C17O, and 17O12C18O in a carbon dioxide sample enriched with oxygen-17 have been recorded with a resolution of about 0.0025 cm-1 in the regions of the fundamental bands, nu2 (600-800 cm-1) and nu3 (2200-2400 cm-1), and in the region of the "forbidden" band, nu1 (1200-1400 cm-1), using the long path difference Fourier transform spectrometer of the LPMA in Paris. For each species, the first hot band in the 4.5-μm region and two hot bands at least in the 15-μm region have been studied for the first time, and a simultaneous reduction of wavenumbers measured in different spectral regions has been carried out yielding new or improved spectroscopic constants. Line intensities have been measured in the region of the nu2 and nu3 bands of 16O12C17O, and the corresponding rotationless transition dipole moments and Herman-Wallis coefficients have been reported. Copyright 1998 Academic Press.     

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.