Building a PCU from the ground up.
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Cardiomyopathy, Dilated↗
Biomedical subjects
M Lutz
Publications and source records attributed to M Lutz.
Characterization of a mutated rubredoxin with a cysteine ligand of the iron replaced by serine.
The active site of rubredoxins consists of a single iron tetrahedrally coordinated to four cysteinate sulfurs. One of the iron ligands, cysteine 42, has been mutated into serine in Clostridium pasteurianum rubredoxin. This mutation resulted in a shift to higher energy of the 320-800 nm region of the UV-visible absorption spectrum. Resonance Raman spectra showed that the nu 1 breathing mode of the iron chromophore was upshifted as a result of the C42S mutation. The spectral pattern, however, was not largely disturbed by the mutation. The EPR spectra of both the wild type and the C42S mutated protein displayed the characteristic features, at g = 4.3 and g = 9.5, of the "3/2" and "1/2" Kramers' doublets, respectively, of a S = 5/2 multiplet. These combined data afford strong evidence that in the C42S mutated rubredoxin serine has replaced cysteine 42 as a ligand of the iron, while maintaining the tetrahedral coordination of the metal. The most spectacular effect of the C42S mutation was a ca. 200 mV downshift of the redox potential of rubredoxin.
Base Sequence↗
On the importance of the "antagonist assumption" to how receptors express themselves.
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Animals↗
Tandem bullet versus multiple gunshot wounds.
The differentiation of tandem bullets fired simultaneously versus multiple bullets fired separately entering through a single entrance wound may present difficulty in wound interpretation for the forensic pathologist. The authors present a case report of three separate projectiles entering through a single perforation. The differentiation of projectiles fired in tandem and multiple single projectiles is discussed.
Brain Injuries↗
Mutated forms of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster.
The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60). Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster. Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented. All of the cysteine residues have individually been replaced by serine and two of them by alanine. Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the [2Fe-2S] cluster. The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated [2Fe-2S] clusters. The C11S-mutated protein was considerably less stable than the wild type ferredoxin. This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the [2Fe-2S] cluster. Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties. Thus, either cysteine 24 is not a ligand of the [2Fe-2S] cluster or it is replaced by another ligand in the C24A mutated protein. A [2Fe-2S] cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off
Alanine↗
Structure and interactions of phycocyanobilin chromophores in phycocyanin and allophycocyanin from an analysis of their resonance raman spectra.
Raman spectra of phycocyanobilin, phycocyanin, and allophycocyanin were obtained at resonance with their visible and near-UV transitions. These spectra were empirically assigned with the help of 14N- and 15N-isotopic substitutions and comparisons with resonance Raman spectra of phycoerythrin. These results confirm the previously suggested assignment of a conformation-sensitive band around 1239-1246 cm-1 to a mode involving nu CmH and nu CN coordinates. Computer-assisted decomposition of the complex, conformation-sensitive 1580-1670-cm-1 region yielded five components that we labeled I-V. The previously described spectral changes observed upon monomerization and denaturation in resonance Raman spectra of phycocyanin and allophycocyanin essentially arise from changes in the relative intensities of these components. Component I (around 1649-1651 cm-1) and component III (1621-1624 cm-1) originate predominantly from nu C=C at C15 of the chromophore. Their relative intensity ratio reflects the relative amounts of C15-Z-anti and C15-Z-syn methine bridge conformations, respectively. Component II (1633-1638 cm-1) is ascribed to a nu C=C mode of pyrrole rings; it is not sensitive to the chromophore conformation. Component IV is also conformation-insensitive and originates from nu C=N and nu C=C coordinates, most likely from ring C. Component V (1591-1594 cm-1) involves a nu C=N coordinate in ring D, coupled to a nu C=C coordinate of the C15 methine bridge. The implications of the present assignments on those of resonance Raman active modes of phytochrome are discussed. A consistent set of correlations between chromophore conformations and resonance Raman data is obtained for both phycobiliproteins and phytochrome.
Cyanobacteria↗
Structure and binding site of the primary electron acceptor in the reaction center of Chlorobium.
In isolated, chlorosome-free reaction centers from Chlorobium limicola f thiosulphatophilum, a chlorin pigment exhibits a Qy absorption band at 672 nm (Feiler, U., Nitschke, W., & Michel, H. (1992) Biochemistry 31, 2608-2614). To characterize the chemical nature of this chlorin pigment and its interactions within the reaction-center protein, selective enhancement of its Raman scattering was achieved by resonant excitation within its Soret band. This is the first time that structural studies of this pigment were performed on the native reaction-center protein. The obtained resonance Raman spectra were consistent with a single population of a chlorophyll a(-like) pigment, possessing a vinyl group on ring I, but not with bacteriochlorophyll c or bacteriophaeophytin c. The stretching frequencies of the C9-keto carbonyl of this pigment indicates that it is H-bonded to the reaction-center protein. The strength of this H-bond is very close to those of the keto carbonyls of the primary electron acceptors in purple bacterial reaction centers and D1/D2 particles. Since in membranes of Chlorobiaceae a transient bleaching at 670 nm is due to the primary acceptor in the reaction center (Nuijs, A. M., Vasmel, H., Joppe, H. L. P., Duysens, L. N. M., & Amesz, J. (1985a) Biochim. Biophys. Acta 907, 24-34), we thus conclude that the primary acceptor in Chlorobium reaction centers is the characterized chlorophyll a(-like) pigment.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacteria↗
Mechanisms of heterologous agonist-stimulated phosphorylation of cholecystokinin receptor.
The phosphorylation of one receptor that occurs as a result of the stimulation of a different receptor on a cell is a common mechanism for heterologous regulation or "cross-talk," which has been implicated in desensitization. In this work, we focus on the mechanisms of phosphorylation of the rat pancreatic acinar cell cholecystokinin (CCK) receptor that occur upon stimulation of this cell by various agonists. Phosphorylation was allowed to occur in dispersed intact acinar cells in response to the experimental manipulation, and the phosphoreceptor was subsequently purified and quantified as an indication of response. Agonists such as vasoactive intestinal polypeptide and secretin, which act via activation of adenylate cyclase, had no effect on CCK receptor phosphorylation, whereas carbamylcholine and bombesin stimulated increased phosphorylation of the CCK receptor. Because these agents would be expected to activate protein kinase C (PKC) as well as a number of calcium-sensitive kinases and phosphatases, these activities were further dissociated by using more direct activators and inhibitors acting intracellularly. Manipulation of calcium independent of PKC by using a calcium ionophore, inhibition of calcium/calmodulin-dependent kinase II, and inhibition of calcium-dependent protein phosphatase type 2B had no effect on the state of CCK receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl Cyclases↗
Structure, spectroscopic, and redox properties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor.
Single mutations of three amino acid residues in the vicinity of the primary electron donor, P, in the reaction center (RC) from Rhodobacter (Rb.) sphaeroides were constructed and characterized in order to study the effects of hydrogen-bonding on the physical properties of P. The mutations, Phe M197-->Tyr, Met L248-->Thr, and Ser L244-->Gly, represent single amino acid changes near P designed to introduce residues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the effects of nonconserved residues. The mutations were designed to change the nonconserved H-bonding interactions of P in Rb. sphaeroides, at the level of a C2 acetyl, a C9 keto, and a C10 ester carbonyl of P, respectively, to those present in Rps. viridis. The Fourier transform (pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized states, from reaction centers of these mutants were studied to determine modifications of H-bond interactions of the pi-conjugated C2 acetyl and C9 keto carbonyl groups and the C10 carbomethoxy ester carbonyl groups of P. The vibrational spectra of reduced P in the Met L248-->Thr and Ser L244-->Gly mutants reveal no evidence for changes in the H-bonding pattern of P; this suggests that for Rb. sphaeroides wild type, Ser L244 is not H-bonded to the C10 ester carbonyl of PL. The vibrational spectrum of reduced P from the Phe M197-->Tyr mutant compared to that of wild type can unambiguously be interpreted in terms of the formation of a new H-bond with an acetyl carbonyl of P, specifically PM. Correlating with the new H-bond, the Phe M197-->Tyr mutant exhibits an electronic absorption spectrum where the P absorption band is significantly perturbed. Intact cell and chromatophore photobleaching spectra of the same mutant indicate that the P absorption band has red-shifted by ca. 10 nm; no such behavior is observed for the other mutants. As well, the P-->BPheL electron transfer rate does not seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of PM to a tyrosine residue. The EPR zero-field splitting parameters, E and D, of the primary donor triplet are only slightly modified in the mutant reaction centers, on the order of 1%.(ABSTRACT TRUNCATED AT 400 WORDS)
Bacteriochlorophylls↗
Comparative effects of rose hip and corn oils on biliary and plasma lipids in rats.
The comparative effects of dietary level and time of feeding corn (CO) and rose hip (RHO) oils on bile and plasma lipid composition were studied. 48 males Sprague Dawley rats were divided in two groups fed semipurified diets containing CO or RHO as the only lipid source. Groups of 6 rats were fed ad libitum diets containing 5% or 15% vegetable oil during 15 or 60 days. Food intake was not dependent on the type of oil, and was higher in 15% oil diets (p < 0.01), increasing with time of feeding (p < 0.001). Bile flow was similar in all groups. Biliary concentrations of cholesterol, phospholipids and bile acids were affected by the time of feeding (p < 0.001). Plasma total and high density lipoprotein (HDL) cholesterol levels were higher in 15% oil fed rats (p < 0.05). Triglycerides concentrations were similar in all groups. The results indicate that oil concentration and time of feeding were the most important variables affecting the lipid composition of rats, independently of the fatty acid composition of the ingested fats.
Animals↗
Anti-CD2 antibodies induce T cell unresponsiveness in vivo.
The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.
Animals↗
Primary donor structure and interactions in bacterial reaction centers from near-infrared Fourier transform resonance Raman spectroscopy.
Preresonance Raman and resonance Raman spectra of the primary donor (P) from reaction centers of the Rhodobacter (Rb.) sphaeroides R26 carotenoidless strain in the P and P+ states, respectively, were obtained at room temperature with 1064-nm excitation and a Fourier transform spectrometer. These spectra clearly indicate that the chromophore modes are observable over those of the protein with no signs of interference below 1800 cm-1. The chromophore modes are dominated by those of the bacteriochlorophylls (BChl a), and it is estimated that, in the P state, ca. 65% of the Raman intensity of the BChl a modes arises from the primary donor. This permits the direct observation of a vibrational spectrum of the primary donor at preresonance with the excitonic 865-nm band. The Raman spectrum of oxidized reaction centers in the presence of ferricyanide clearly exhibits bands arising from a BChl a+ species. The magnitude of the frequency shift of a keto carbonyl of neutral P from 1691 to 1717 cm-1 upon P+ formation strongly suggests that one BChl molecule in P+ carries nearly the full +1 charge. Our results indicate that the unpaired electron in P.+ does not share a molecular orbital common to the two components of the dimer on the time scale of the resonance Raman effect (ca. 10(-13) s).
Fourier Analysis↗
Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210.
Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.
Kinetics↗
In vitro studies on drug interaction of ifosfamide and ACNU in primary and metastatic human brain tumours.
Combination chemotherapy is widely employed in clinical oncology; however, there is no generally accepted model to evaluate individual tumour susceptibility to a given drug combination protocol. We therefore investigated the drug interaction of ifosfamide (4-hydroxyperoxy-ifosfamide) and ACNU in a recently developed in vitro model of paired sequential combination chemotherapy in primary and metastatic malignant brain tumours. A long-term standard [6,3-3H]-thymidine-incorporation assay, employing a liquid scintillation counting protocol, was selected to assess the drug sensitivity of human tumours. In vitro drug exposures were derived from correlating in vivo-(systemic and CNS) and in vitro-pharmacokinetic drug parameters. In combination experiments tumour cells were treated sequentially by two drugs in both sequences: drug exposures were calculated for 2 h with a 1-h drug-free interval in between. "Cut-off" concentrations (maximum in vitro exposure doses) were calculated as 1.74 microM (for primary CNS tumours: 0.58 microM) for ifosfamide and 5.4 microM (for primary CNS tumours: 1.33 microM) for ACNU. Dose/response relations were derived from isotope incorporation rates after cells had grown for approximately five population doubling times. Combination isoboles were plotted after drug doses had been transformed into "equieffective doses", enabling comparison of drug combination effects. In all three glioblastomas (with CNS exposure dose) an additive or supra-additive effect could be observed in either sequence (in one tumour a biphasic additive isobole was found for both sequences). Out of three bronchial carcinomas (small-cell type, brain metastases) in two non-identical sequences a supra-additive effect was observed in two tumours, with antagonistic effects in the third tumour. In all three malignant melanomas and in one renal carcinoma antagonistic effects were observed, whereas in a second renal carcinoma supra-additive effects were demonstrated for both sequences. We conclude that drug combination chemotherapy effects at the cellular level may be extremely heterogeneous.
Antineoplastic Combined Chemotherapy Protocols↗
Structure of the primary electron donor in photosystem I: a resonance Raman study.
Low-temperature resonance Raman (RR) spectra have been obtained at resonance with the Soret transition of chlorophyll a in photosystem I particles containing large amounts either of the triplet state of P700 or of its radical cation state. Subtracting these spectra from those of resting reaction centers yielded RR spectra of P700 in its neutral, ground state. These spectra arise from two distinct chlorophyll a molecules differing by the strengths of the bonding interactions assumed by their keto carbonyl groups, the stretching frequencies of which are found at 1655 and 1675 cm-1. The present results rule out previous hypotheses that P700 might have consisted of a single, chemically modified chlorophyll a molecule. Neither of the bonding interactions assumed by the keto carbonyls of the P700 chlorophylls most probably involves chlorophyll-chlorophyll bridging through water molecules, as surmised in the so-called special pair models, but likely consists of H bonds with distinct protein sites. The magnesium atoms of the two P700 chlorophylls are 5-coordinated. Hence, the structural model of P700 provided by the present data is qualitatively the same, in terms of bonding interactions, as that currently accepted for the bacterial primary donor.
Binding Sites↗
Histologic sampling requirements in ovarian carcinoma: a review of 51 tumors.
In a review of 29 serous and 22 mucinous adenocarcinomas of the ovary, we quantitated the various histologic patterns seen in these neoplasms in order to document histologic variability and assess current anecdotal recommendations (one section per 1-2 cm of maximum tumor diameter) for adequate histologic sampling of epithelial ovarian neoplasms. Mucinous carcinomas showed more histologic variation than serous carcinomas, but both showed substantial portions with benign or borderline histology in many neoplasms. One serous and one mucinous carcinoma were likely to have been classified as borderline tumors if only current sampling recommendations had been followed. The remaining adenocarcinomas were adequately sampled by current standard practice. The two tumors in question had unusual clinical and histological features which prompted additional sampling. Caution is particularly warranted when initial frozen section sampling shows features of borderline tumor histology or low grade epithelial atypia, especially in mucinous epithelial neoplasms where histologic variability is greatest.
Adenocarcinoma, Mucinous↗
Drug and tissue factors both confound KA measurements.
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Receptors, Drug↗
Occlusive vascular stapling in a canine inferior vena cava model.
A canine model for inferior vena cava resection was devised to study the biomechanical and clinical applications of the cartridge stapler, which applies a triple staggered staple line. Eighteen mongrel dogs were subjected to subhepatic, suprarenal inferior vena cava occlusion with excision of the excluded segment. At 1 hour, six dogs were killed and inferior vena cava sizes and pressures were measured. All staple lines were intact. The inferior vena cava bursting pressure was 150 to 300 mm Hg. Ten dogs survived to 6 weeks and underwent repeat laparotomy with excision and measurement of the remaining inferior vena cava. Mean diameter was 1.9 cm. No evidence of staple line leakage was found despite chronic pressures to 39 cm H2O: median static bursting pressures were 250 mm Hg. Vein wall thickness of greater than 0.24 mm were all satisfactorily stapled. Histologic examination revealed normal reendothelialization, with improved healing at higher chronic pressures. No evidence of inflammation at the staple line or foreign body reaction was found. We conclude that a triple staple line provides secure, rapid venous occlusion that can withstand supraphysiologic stresses immediately and at 6 weeks.
Animals↗