C2 Complex Research Articles

Placement of C2 laminar screws using three-dimensional fluoroscopy-based image guidance by Eric W. Nottmeier and Andrew B. Foy

In their work colleagues Nottmeier and Foy present a retrospective series of eight patients treated with posterior C1–C2 fusion by laminar screws using three-dimensional fluoroscopy-based image guidance. They conclude from the results of their work that: “In this study, it has been shown that C2 laminar screws can be safely and accurately placed using 3D fluoroscopy-based image guidance. Advantages of this technology over traditional techniques include real-time 3D computerized feedback to the surgeon during placement, as well as the ability to size the screw to the patient’s anatomy using intraoperative computerized planning.” In the last decade, technology has advanced to the point where regular computer workstations are able to calculate in real-time the transformation matrices needed to determine the position of a camera-tracked object (in a surgical setting, typically a frame with fiducials) in relation to a camera-tracked reference base. By attaching the reference base to a patient and (surgical) instruments to camera-tracked objects, the workstation can calculate the respective positions/orientations and display their virtual interactions. When three-dimensional data sets representing the patient’s anatomy from modalities including CT/MRI/3D rotational X-ray imaging, are manipulated so that virtual and real reference bases are exactly aligned, surgical instruments and/or implants can be displayed on anatomical structures not visible for the surgeon intraoperatively. This is, in a nutshell, current “computer-assisted surgery” or “image-guided surgery”. Assuming that transformation matrix calculations are mathematical processes carried out with high fidelity and great reproducibility, imperfections in navigational accuracy must have other sources. Calibration errors in 3D fluoroscopy-based systems have, for example, been shown to account for approximately 1 mm accuracy in a phantom model [1]. In a more realistic cadaveric model, the accuracy of navigation deteriorated to approximately 2.5 mm, mainly due to bending of instruments and/or references during manipulation [2]. In clinical practice more inaccuracies can be introduced by occasional blocking of the camera field of view; inadvertently touching/hitting references and, especially, a nonrigid connection between the patient reference base and the actual surgical site. In the present study, the patient reference base was attached to a head clamp, which was, in turn, connected via pins to the patient’s skull while surgery was performed at the levels C1 and C2. This experimental setup represents quite a long chain of connections from the reference base to the surgical site with considerable opportunity for movement between the two extremes. This is without taking into account additional movements due to, for example, patient ventilation and direct manipulation of C1 and C2 by surgical instruments. Considering that the C0–C1–C2 complex is highly mobile, it came as a small surprise for this reviewer to find such accurate screw placement in the current study. As can be found in the article, the investigators did verify the image-guided technique (“After accuracy of navigation was confirmed by the surgeon ... ”). The interesting question that follows automatically is: “How?” Checking accuracy by probing the patient reference base with a navigated instrument and finding a perfectly matched position/orientation on the computer display does nothing for the flexibility/movement errors that may be present at the more distant surgical site. Most often, surgeons verify accuracy by a direct intraoperative view of the surgical site to see whether, for example, the proposed drill trajectory or planned screw length is in accordance to their own experience from conventional surgeries before moving on with navigation. This leads to the strange paradox that is really bugging contemporary image-guided procedures: the employment of a technique to enhance surgical accuracy requiring the surgeon’s intraoperative verification of its accuracy. The reluctance of surgeons to show complete faith in image guidance seems justified considering the errors demonstrated in accuracy studies. Using computer navigation without proper anatomical and surgical knowledge would be irresponsible. Armed with proper knowledge, however, navigation becomes largely superfluous since surgeons trust their surgical experience/skills and the direct intraoperative view over computer-generated images anytime, and adjust their actions accordingly. Which surgeon would happily volunteer when asked to perform a percutaneous procedure having to trust solely on information provided by any image-guided technique commercially available today? Until now, no large-scale long-term and properly controlled study has been published in the field of computer-assisted surgery that has convincingly showed any actual patient benefit when using image guidance. Lacking a control group and using a navigation technique susceptible to the aforementioned accuracy problems, the present study may, unfortunately, not be able to change this status.

Poster 282: Unstable Jefferson Fracture as an Etiology for Persistent Dysphagia and Dysphasia in a Tetraplegic Patient: A Case Report

Setting: University hospital, rehabilitation ward. Patient: A 37-year-old C4 tetraplegic, 1 month postinjury, with persistent dysphagia and dysphasia. Case Description: The patient was admitted following a motorcycle crash, with resultant C4 complete tetraplegia. Immediately postinjury, prior to intubation, the patient was vocalizing. Injuries included: stable Jefferson (C1) fracture, C2 complex fracture, C5 fracture and small frontal subdural hematoma. Operative management included C5 corpectomy and C4-7 anteroposterior fusion.

Cytochrome c2Exit Strategy: Dissociation Studies and Evolutionary Implications

Small, water-soluble, type c cytochromes form a transient network connecting major bioenergetic membrane protein complexes in both photosynthesis and respiration. In the photosynthesis cycle of Rhodobacter sphaeroides, cytochrome c2 (cyt c2) docks to the reaction center (RC), undergoes electron transfer, and exits for the cytochrome bc1 complex. Translations of cyt c2 about the RC-cyt c2 docking interface and surrounding membrane reveal possible exit pathways. A pathway at a minimal elevation allowed by the architecture of the RC is analyzed using both an all-atom steered molecular dynamics simulation of the RC-cyt c2 complex and a bioinformatic analysis of the structures and sequences of cyt c. The structure-based phylogenetic analysis allows for the identification of structural elements that have evolved to satisfy the requirements of having multiple functional partners. The patterns of evolutionary variation obtained from the phylogenetic analysis of both docking partners of cyt c2 reveal conservation of key residues involved in the interaction interfaces that would be candidates for further experimental studies. Additionally, using the molecular mechanics Poisson-Boltzmann surface area method we calculate that the binding free energy of reduced cyt c2 to the RC is nearly 6 kcal/mol more favorable than with oxidized cyt c2. The redox-dependent variations lead to changes in structural flexibility, behavior of the interfacial water molecules, and eventually changes in the binding free energy of the complex.

Role of Water in Transient Cytochrome c2 Docking

Cytochrome c (cyt c) is a small water-soluble redox protein that facilitates electron transfer in photosynthesis and respiration by alternately docking to integral membrane proteins such as the photosynthetic reaction center (RC). Recently, a high-resolution X-ray structure was solved for the RC−cyt c2 complex of Rhodobacter sphaeroides, revealing important contacts between the RC in its ground state and reduced cyt c2 mediated by bridging water molecules. In this article, we compare the variations in these contacts and in the interface in general for both redox states of cyt c2 that resulted from full-atom simulations of the complexes embedded in a membrane with explicit water molecules. Molecular dynamics simulations of the two redox states of the RC−cyt c2 system were performed using the CHARMM27 parameters developed for the oxidized and reduced forms of the heme prosthethic group. In its overall dynamics, the encounter complex was found to be very similar in both redox states, exhibiting at the interface a stable hydrophobic tunneling domain and a broad basin of attraction. The differences between the redox states are subtle and involve the formation of a structured cluster of water molecules in the reduced cyt c2 system. Fluctuations of water and residues at the interface increase upon oxidation and probably mediate the undocking process. The observed differences between the two redox states of the system can only be attributed to the different electrostatic potentials generated by heme in the interface region, as no other modifications were introduced. As the time scale of the undocking process is beyond the time scales reachable by full atomic molecular dynamics simulation of the system, we employed steered molecular dynamics to investigate and compare the energetics associated with the unbinding of RC−cyt c2 in the reduced and oxidized forms.

Variable LH2 stoichiometry and core clustering in native membranes of Rhodospirillum photometricum.

The individual components of the photosynthetic unit (PSU), the light-harvesting complexes (LH2 and LH1) and the reaction center (RC), are structurally and functionally known in great detail. An important current challenge is the study of their assembly within native membranes. Here, we present AFM topographs at 12 A resolution of native membranes containing all constituents of the PSU from Rhodospirillum photometricum. Besides the major technical advance represented by the acquisition of such highly resolved data of a complex membrane, the images give new insights into the organization of this energy generating apparatus in Rsp. photometricum: (i) there is a variable stoichiometry of LH2, (ii) the RC is completely encircled by a closed LH1 assembly, (iii) the LH1 assembly around the RC forms an ellipse, (iv) the PSU proteins cluster together segregating out of protein free lipid bilayers, (v) core complexes cluster although enough LH2 are present to prevent core-core contacts, and (vi) there is no cytochrome bc1 complex visible in close proximity to the RCs. The functional significance of all these findings is discussed.

Progesterone receptor activates its promoter activity in human endometrial stromal cells

Previous studies have shown that progestin increases the content of progesterone receptor (hPRA and hPRB) and the hPR mRNA during decidualization of human endometrial stromal cells suggesting that endogenous hPR enhances the transcription of the hPR gene. In the present study, we provide evidence that hPR regulates the promoter activity mediated through an active Sp1 site. In stromal cells treated with medroxyprogesterone acetate, the promoter activity was significantly increased when cells were co-transfected with hPR expression vector. Progressive deletion analysis showed that the highest activity was derived from the promoter region between −55 and +31 bp. Transactivation by hPR was dose dependent. The capacity of hPRA was stronger than that of hPRB. The ligand binding domain, but not DNA binding domain of the hPR was required for the transactivation. The proximal promoter region lacks a canonical progesterone response element. Instead, an active Sp1 site (−49 to −43 bp) has been confirmed. Mutation of the Sp1 site eliminated the effect of hPR activation. The promoter activity was increased by over expression of Sp1, whereas Sp3 had no effect. Electrophoretic mobility shift assay showed that the promoter region between −55 and +31 bp bound to Sp1 family proteins, Sp1 (C2 complex) and Sp3 (C1 and C3 complexes) identified by antibodies to Sp1 and Sp3. Sp1 complex formed by extracts of stromal cells was less intense than that formed by progestin-decidualized stromal cells. Sp1/DNA binding was enhanced when stromal cell extracts were incubated with calf intestine alkaline phosphatase (CIP) suggesting that dephosphorylation of Sp1 enhances the DNA binding. Addition of protein kinase inhibitor, H-89 or H-7, enhanced the hPR stimulated promoter activity. Western blot analysis showed that endometrial stromal/decidual cell extracts contained a wide band of Sp1 spanning from ∼105 to 96 kDa and was resolved into one band at 96 kDa by CIP. Decidual cell extracts are abundant with the 96 kDa Sp1. In addition, the 96 kDa Sp1 was co-precipitated with ligand-activated hPRA or hPRB in the decidual cell nuclear extracts. These data suggest that dephosphorylated Sp1, abundant in decidual cells, enhances the binding to both DNA and hPR resulting in a robust increase of the hPR promoter activity.

Posterior C1-C2 fusion with polyaxial screw and rod fixation.

A novel technique of atlantoaxial stabilization using individual fixation of the C1 lateral mass and the C2 pedicle with minipolyaxial screws and rods is described. In addition, the initial results of this technique on 37 patients are described. To describe the technique and the initial clinical and radiographic results for posterior C1-C2 fixation with a new implant system. Stabilization of the atlantoaxial complex is a challenging procedure because of the unique anatomy of this region. Fixation by transarticular screws combined with posterior wiring and structural bone grafting leads to excellent fusion rates. The technique is technically demanding and has a potential risk of injury to the vertebral artery. In addition, this procedure cannot be used in the presence of fixed subluxation of C1 on C2 and in the case of an aberrant path of the vertebral artery. To address these limitations, a new technique of C1-C2 fixation has been developed: bilateral insertion of polyaxial-head screws in the lateral mass of C1 and through the pars interarticularis into the pedicle of C2, followed by a fluoroscopically controlled reduction maneuver and rod fixation. After posterior exposure of the C1-C2 complex, the 3.5-mm polyaxial screws are inserted in the lateral masses of C1. Two polyaxial screws are then inserted into the pars interarticularis of C2. Drilling is guided by anatomic landmarks and fluoroscopy. If necessary, reduction of C1 onto C2 can be accomplished by manipulation of the implants, followed by fixation to the 3-mm rod. For definitive fusion, cancellous bone can be added. No structural bone graft or wiring is required. In selected cases, e.g., C1-C2 subluxation or fractures in young patients in whom only temporary fixation is necessary, the instrumentation can be removed after an appropriate time. Because the joint surfaces stay intact, the patient can regain motion in the C1-C2 joints. Thirty-seven patients underwent this procedure. No neural or vascular damage related to this technique has been observed. The early clinical and radiologic follow-up data indicate solid fusion in all patients. Fixation of the atlantoaxial complex using polyaxial-head screws and rods seems to be a reliable technique and should be considered an efficient alternative to the previously reported techniques.

Lipase Production in Two-Step Fed-Batch Culture of Organic Solvent-Tolerant Pseudomonas aeruginosa LST-03.

Efficient lipase production by two-step fed-batch culture of an organic solvent-tolerant bacterium, Pseudomonas aeruginosa LST-03, was investigated. When FB synthetic medium was used in flask culture, no lipase activity was detected, whereas lipase was produced at 2.3 I.U./ml in C2 complex medium. However, lipase production was induced in FB medium when a fatty acid was added to the culture broth in the stationary phase. Among fatty acids tested, long chain saturated fatty acids, such as C18 (stearic acid) and C20 (arachidic acid), were found to function as effective inducers for the production of lipase, giving an activity level almost the same as that obtained in C2 medium in flask culture. Two-step lipase production, comprised of a growth phase in fed-batch mode and a production phase in which lipase was induced by the addition of 5% (v/v) stearic acid, was carried out in a jar-fermentor. In the growth phase, the maximum cell concentration at 16 h was only 20 in terms of the optical density at 660 nm (OD660), and a low level of lipase production (8 I.U./ml) was obtained after 167 h. This was considered to be due to the exhaustion of several medium components brought about by the use of an unsuitable medium or feeding solution. After analyzing the contents of the compounds in the culture broth by inductively coupled plasma spectrometry for metal ions and HPLC for anions, a modified FB medium was designed. When this modified FB medium was used in two-step fed-batch culture, the maximum cell concentration reached an OD660 of 55 (30.2 g-dry cells/l) at 16.5 h, and lipase was produced at 96 I.U./ml after 35 h, which is approximately 40 times higher than the production level obtained in flask culture using C2 medium.

Complement C4bC2 complex formation: an investigation by surface plasmon resonance

Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30–830 mM NaCl) and in the absence and presence of MgCl 2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg 2+ in the range of 2.5–15 mM. C4bC2 affinity ( K d) was determined by steady-state analysis to be 7.2×10 −8 M in physiological conditions (10 mM Hepes, 2.5 mM MgCl 2, 0.75 mM CaCl 2 and 140 mM NaCl, pH 7.4). For C4(H 2O)C2 complex formation, a K d of 4.0×10 −8 M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg 2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. K d was 1.5×10 −8 M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500–1000 nM). K d was 1.2×10 −6 M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.

Lipase production in two-step fed-batch culture of organic solvent-tolerant Pseudomonas aeruginosa LST-03

Efficient lipase production by two-step fed-batch culture of an organic solvent-tolerant bacterium, Pseudomonas aeruginosa LST-03, was investigated. When FB synthetic medium was used in flask culture, no lipase activity was detected, whereas lipase was produced at 2.3 I.U./ml in C2 complex medium. However, lipase production was induced in FB medium when a fatty acid was added to the culture broth in the stationary phase. Among fatty acids tested, long chain saturated fatty acids, such as C18 (stearic acid) and C20 (arachidic acid), were found to function as effective inducers for the production of lipase, giving an activity level almost the same as that obtained in C2 medium in flask culture. Two-step lipase production, comprised of a growth phase in fed-batch mode and a production phase in which lipase was induced by the addition of 5% (v/v) stearic acid, was carried out in a jar-fermentor. In the growth phase, the maximum cell concentration at 16 h was only 20 in terms of the optical density at 660 nm (OD 660), and a low level of lipase production (8 I.U./ml) was obtained after 167 h. This was considered to be due to the exhaustion of several medium components brought about by the use of an unsuitable medium or feeding solution. After analyzing the contents of the compounds in the culture broth by inductively coupled plasma spectrometry for metal ions and HPLC for anions, a modified FB medium was designed. When this modified FB medium was used in two-step fed-batch culture, the maximum cell concentration reached an OD 660 of 55 (30.2 g-dry cells/ l) at 16.5 h, and lipase was produced at 96 I.U./ml after 35 h, which is approximately 40 times higher than the production level obtained in flask culture using C2 medium.

Identification of a Novel Cytokine Response Element in the Human IFN Regulatory Factor-1 Gene Promoter

The present study investigates the regulatory mechanisms involved in the cooperation between IFN-gamma and TNF-alpha to promote transcription from IFN regulatory factor-1 (IRF-1). A transient transfection analysis revealed that the region between -218 and -144, where +1 is the transcription start site, as well as previously reported downstream elements, ppkappaB and IFN-gamma activation site/kappaB, were required for the optimal response to the two cytokines. A subsequent DNase I footprint analysis showed that the region between -171 and -144 was inducibly protected with stimulation by TNF-alpha, and this protection was significantly enhanced with the combination of IFN-gamma and TNF-alpha. In an EMSA with the protected region as a probe, a TNF-alpha-inducible complex (C1) and an IFN-gamma-inducible complex (C2), but no synergy-specific DNA-protein complexes, were recognized. The C1 complex consisted of a pre-existing factor (p65/p50), whereas the C2 complex consisted of a newly synthesized IRF-1-related factor. A methylation interference assay revealed the critical G residues (from -167 to -151) for the DNA-protein complex formation specific to the cytokine response, and within this region the novel kappaB sequence, the promoter distal kappaB (pdkappaB) element (5'-GGGGAAG TAC-3'), was identified. Because the base substitutions over the pdkappaB region (from -171 to -144) affected not only the TNF-alpha-response but also that of IFN-gamma, this region might contribute to the cooperative action of the NF-kappaB subunits with the IRF-1-related factor. Finally, we demonstrated that none of the cis-acting elements, ppkappaB, pdkappaB, or IFN-gamma activation site/kappaB, is dispensable for the optimal synergism in response to IFN-gamma and TNF-alpha.

Distinct Ca2+ Binding Properties of Novel C2 Domains of Plant Phospholipase Dα and β

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDalpha requires millimolar levels of Ca(2+) for optimal activity, whereas PLDbeta is most active at micromolar concentrations of Ca(2+). Multiple amino acid sequence alignments suggest that PLDalpha and PLDbeta both contain a Ca(2+)-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDalpha and PLDbeta, designated PLDalpha C2 and PLDbeta C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major beta-sheet structures but underwent spectral changes upon binding Ca(2+); the magnitude was larger for PLDbeta C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca(2+) concentrations for PLDbeta C2 but at millimolar levels of the cation for PLDalpha C2. PLDbeta C2 exhibited three Ca(2+)-binding sites: one with a dissociation constant (K(d)) of 0.8 microm and the other two with a K(d) of 24 micrometer. In contrast, isothermal titration calorimetry data of PLDalpha C2 were consistent with 1-3 low affinity Ca(2+)-binding sites with K(d) in the range of 590-470 micrometer. The thermodynamics of Ca(2+) binding markedly differed for the two C2 domains. Likewise, PLDbeta C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca(2+) concentrations, whereas PLDalpha C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca(2+) displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca(2+) and PC binding properties of PLDalpha C2 and PLDbeta C2 follow a trend similar to the Ca(2+) requirements of the whole enzymes, PLDalpha and PLDbeta, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDalpha and PLDbeta have novel structural features and serve as handles by which Ca(2+) differentially regulates the activities of the isoforms.

Protein Dynamics: Imidazole Binding to Class I C-Type Cytochromes

The oxidized cytochrome c2 from the purple phototrophic bacteria, Rhodobacter sphaeroides and Rhodobacter capsulatus, bind the neutral species of imidazole (Ka = 1440 ± 40 M−1) 50 times more strongly than does horse mitochondrial cytochrome c (Ka = 30 ± 1 M−1). The kinetics of imidazole binding are consistent with a change in rate-limiting step at high ligand concentrations for all three proteins. This is attributed to a conformational change leading to breakage of the iron–methionine bond which precedes imidazole binding. The three-dimensional structure of the Rb. sphaeroides cytochrome c2 imidazole complex (Axelrod et al., Acta Crystalogr. D50, 596–602) supports the view that the conformational changes are essentially localized to approximately seven residues on either side of the ligated methionine and there is a hydrogen bond between the Phe 102 carbonyl, an internal water, and the bound imidazole. Insertions and deletions in this region of cytochrome c2, the presence of a proline near the methionine, and the smaller size of the dynamic region of horse cytochrome c suggest that the stabilizing hydrogen bond is not present in horse cytochrome c, hence, the dramatic difference in affinity for imidazole. The kinetics of ligand binding do not correlate with either the strength of the iron–methionine bond as measured by the pK of the 695-nm absorption band or the overall stability of the cytochromes studied. However, the very similar imidazole binding properties of the two cytochromes c2 indicate that the Rb. sphaeroides cytochrome c2–imidazole complex structure is an excellent model for the corresponding Rb. capsulatus cytochrome c2 complex. It is notable that the movement of the peptide chain in the vicinity of the ligated methionine has been preserved throughout evolution and suggests a role in the function of c-type cytochromes.

Zonal organization of cortico-nuclear and nucleo-cortical projections of the paramedian lobule of the cat cerebellum. 2. The C 2 zone

The cortico-nuclear (C-N) and nucleo-cortical (N-C) projections of the C2 cortical zone in pars anterior (pa) and pars posterior (pp) of the paramedian lobule (PML) in the posterior lobe of the cat cerebellum were investigated with a combined electrophysiological and neuroanatomical technique. In each experiment the mediolateral boundaries of the zone were localized on the cortical surface by recording field potentials mediated via climbing fibres and evoked in the zone by activity elicited in spino-olivocerebellar paths through percutaneous stimulation of the fore- and hindlimbs; a small (15-30 nl) injection of 1-2% WGA-HRP was then made into the zone. Distributions in the deep cerebellar nuclei were determined with light microscopy both for C-N terminal labelling due to anterograde axonal transport by Purkinje cells and for cell bodies labelled due to retrograde transport in N-C axons. The extent to which retrogradely labelled olivary neurones were confined to the part of the rostral medial accessory olive that innervates the C2 zone was estimated to provide an indication of the degree to which the injected tracer might have spread beyond the boundaries of the zone. The C-N projection from the part of the C2 zone in PML pa terminates almost exclusively (probably exclusively) in nucleus interpositus posterior (NIP) at all medio-lateral levels of the nucleus but most extensively at middle and lateral levels. At most levels the C-N termination territory forms a crescent with its outer curve following the caudal, dorsal and rostral borders of the nucleus and as a result it is mainly in the dorsal half of the nucleus. There is heavy overlap with the projection from the lobule V part of the C2 zone previously studied by us. The projection from the C2 zone in PML pp terminates entirely in NIP, but although at middle medio-lateral levels in the nucleus there is substantial overlap with the PML pa and lobule V projections, the projection territory is confined to the medial half of the nucleus. Evidence was obtained compatible with the view that throughout the C2 zone its lateral and medial parts project to different parts of NIP. In both PML pa and pp the C2 zone receives N-C projections from NIP. Most of the N-C cells concerned are in the dorsal half of NIP and the great majority lie within the corresponding C-N projection territory. However, the N-C projection to PML pa appears c. 6 times heavier than that to PML pp and the PML pa part of the zone also receives a minor additional projection from nucleus lateralis (NL). The findings are discussed in relation to the hypothesis of olivo-cortico-nuclear complexes or compartments, with particular reference to the internal organization of the C2 complex.

SUPRAMOLECULAR ORGANIZATION OF THE PHOTOSYNTHETIC CHAIN IN MUTANTS OF RHODOBACTER CAPSULATUS DELETED IN CYTOCHROME C2

Both the soluble cytochrome c2 and the membrane-bound cytochrome cy act as secondary electron carriers in photoinduced cyclic electron transfer chain of Rhodobacter capsulatus [Jenney and Daldal (1993) EMBO J 12: 1283–1292]. In this work, we have studied the kinetics of electron transfer between these secondary electron donors and the reaction center in intact cells of two mutants, MT-G4/S4 and MT-GS18 deleted in cytochrome c2 and in cytochrome c2 plus cytochrome bc1 complex, respectively. In the MT-G4/S4 mutant, only about one third of the primary electron donor is reduced by cytochrome cy in less than five ms. The remaining fraction is reduced in several seconds, although about 90% of the photoxidized cytochrome cy is reduced in less than 10 ms by the cytochrome bc1 complex. This implies that cytochrome cy is not in thermodynamic equilibrium with the large fraction of primary donors which are slowly reduced. As shown by energy transfer measurements, the reaction centers connected to cytochrome cy and the disconnected reaction centers are localized in the same membrane region. We propose that the movement of cyt cy is restricted to a small membrane domain which includes a single cytochrome bc1 complex. The kinetics of cytochrome cy photooxidation in the MT-G4/S4 mutant in the presence of myxothiazol presents a fast phase (t1/2 ∼ 3 µs) followed by a slower phase (t1/2 ∼ 20 µs). In the case of the double mutant MT-GS18, the kinetics of electron transfer between cytochrome cy and the reaction center is highly multiphasic and much slower than those observed for the MT-G4/S4 mutant. In particular, the amplitude of the fast phase is decreased by more than a factor 2 and the 20-µs phase is not observed. This implies an important structural role of the cytochrome bc1 complex in the interaction between reaction center and cytochrome cy, and their formation in supercomplex. The more problable stoichiometry of electron carriers in this supercomplex is 2 reaction centers, 2 cytochrome cy and 1 cytochrome bc1 complex.

Downregulation of Waf1, C2, C3, and major histocompatibility complex class I loci within an 18-cM region of chromosome 17 in adenovirus-transformed mouse cells.

In this study, the expression of the p53 tumor suppressor gene and the p53-regulated Mdm2 and Waf1 genes was evaluated in adenovirus (Ad)-transformed mouse cells. The expected levels of p53 mRNA and protein and Mdm2 mRNA were detected in all transformed cells. However, the level of Waf1 mRNA was markedly reduced in Ad12-transformed cells and in some Ad5-transformed cells. Waf1 expression was not reduced in untransformed mouse cells infected with Ad12 or Ad5. Expression of the class I major histocompatibility complex (MHC) locus was downregulated in 13 Ad-transformed cell lines (derived from four different strains of mice) that exhibited reduced expression of Waf1. Waf1 is located on mouse chromosome 17 proximal to the MHC class I locus. To determine whether other chromosome 17 genes were downregulated, the cells were examined for expression of other genetic loci. Of those tested, only the C2 and C3 complement loci were expressed in mouse fibroblasts. Expression of C2 (which is within the MHC) and expression of C3 (which is 15 cM distal to the MHC) were downregulated in those transformed cells in which Waf1 and MHC class I were downregulated. The Ad12- and Ad5-transformed cells that expressed low levels of Waf1, MHC class I, C2, and C3 formed tumors in syngeneic adult mice. These data suggest that the downregulation of multiple genes within the 32 Mb of mouse chromosome 17 that includes the Waf1 locus to the C3 locus occurs in Ad mouse-cell transformation and may contribute to the tumorigenicity of transformed cells.

Cross-Linked Electron Transfer Complex between Cytochrome c2 and the Photosynthetic Reaction Center of Rhodobacter sphaeroides

Electron donation from the soluble cytochrome (cyt) c2 to the photooxidized primary donor, P+, of reaction centers isolated from Rhodobacter sphaeroides was studied by using chemical zero-length cross-linking. This cross-linking stabilizes a 1:1 covalent complex between subunit M of the reaction center and cyt c2. In 80% of the reaction centers, P+ generated by a laser flash is reduced by covalently bound cyt c2. Kinetics of P+ reduction show (i) a fast phase with a half-life of 0.7 micros similar to that observed for electron transfer in the noncovalent proximal complex and (ii) a slow phase (t1/2 = 60 micros) that is attributed to a cyt c2 bound less favorably for electron transfer. Its relationship with similar kinetic phases attributed to a distal conformation of the complex in previous studies is discussed. Both kinetic phases are slightly accelerated upon addition of glycerol. Upon addition of reduced soluble cyt c2 to the cross-linked complex the kinetics of both phases are not affected. The kinetics of P+ reduction following the second flash (20 ms after the first) show that a complex is formed between soluble cyt c2 and the cross-linked complex, in which electron transfer takes place in the millisecond time domain. Cross-linked cyt c2 in complexes which give rise to the two kinetic phases of P+ reduction shows almost pH-independent midpoint redox potentials between pH 6 and 9.5. This behavior is at variance with that of free cyt c2, the midpoint potential of which is affected by at least two protonable groups within this pH range. The cross-linked RC-cyt c2 complex allowed study of the effects of temperature on the electron transfer reaction without a possible disturbance by dissociation of the complex. In the 250-300 K range, Arrhenius behavior is observed showing activation energies of 11.7 and 8.0 kJ/mol for the faster and the slower kinetic phases, respectively, which are remarkably lower than the activation energy of 20.5 kJ/mol for the fast P+ reduction by soluble cyt c2 [Venturoli, G., Mallardi, A., & Mathis, P. (1993) Biochemistry 32, 13245-13253]. Between 250 and 230 K, a fall-off in amplitude is observed for both kinetic phases indicating that intracomplex electron transfer is blocked at low temperatures.

Resolution of the Absorbance and CD Spectra and Formation Constants of the Complexes between Human Serum Albumin and Methyl Orange

AbstractDifference absorbance and circular dichroism techniques show that two complexes are formed between human serum albumin (HSA) and Methyl Orange (MO). The stoichiometries of the two HSA‐MO complexes (1:1 (C1) and 1:2 (C2)), their association constants (K1, 1 = 2.32 (0.18) × 105 M−1 and K1, 2 = 1.12 (0.15) × 1011 M−2), and both absorbance and dichroic spectra have been determined by a computational approach. Nearly 900 experimental points, consisting of absorbance and CD measurements registered in the 340–550 nm interval and over a wide range of concentrations of protein and ligand, have been included in a unique fitting procedure. The Scatchard plot indicates the existence of a unique binding site which can accommodate up to two molecules of MO in a positive co‐operative process. Calculation of the CD spectrum for the C2 complex according to the DeVoe method reproduces the fitted dichroic spectrum for the same complex. The shapes of the fitted absorbance and dichroic spectra, as well as the influence of concentrated NaCl or ethylene glycol on the absorbances of both free MO and HSA‐MO mixtures are consistent with the presence of dominant electrostatic interactions in C1. The C2 complex can be envisaged as a unique chromophore, consisting of two MO units associated in a stacking process into the same binding site of HSA, leading to a well‐defined chirality. The general validity of this multitechnique, multiwavelength approach in the investigation of protein‐ligand complexes is discussed.

Evidence for a regulatory protein complex on RNA polymerase III promoter of human retroposons of Alu family.

Abundant human retroposons of the Alu family produce few RNA polymerase III (RPIII)-dependent transcripts in vivo. This suggests that either the bulk of the repeats has no proper promoter elements or that transcription of Alu by RPIII is repressed. In this study, we analyzed complexes formed by human nuclear proteins with the Alu B-box and with an adjacent downstream sequence (DB-sequence). Four complexes (C1-C4) were detected and two of them (C2 and C3) were found to be induced by different proteins. C3 formation was found to be sensitive to minor sequence variation within the Alu DB-sequence. The C2 complex is specifically repressed by the competing VA1 B-box oligonucleotide and was found to be very stable. In addition, it is downregulated in human cells transformed by adenovirus 5. This is consistent with a view that the C2 complex is formed by a protein (designated as ACR1) that is different from TFIIIC2. The ACR1 protein may be involved in the modulation of Alu transcription in vivo by interfering or cooperating with TFIIIC2. A similar complex is detected with the efficiently transcribed adenovirus VA1 RNA gene B-box. We compared the affinity of complexes formed by ACR1 with Alu and VA1 B-boxes. It was found that both B-boxes bind ACR1 with equal affinity with a dissociation constant of about 2 nM. However, DB-sequences in Alu and VA1 promoters are non-homologous, and C3/C4 complexes are found to be formed with Alu DB, but not formed with VA1 DB sequences. The Alu-specific protein forming C3 (named as ACR2) may cooperate with ACR1 in selective repression of RPIII-dependent Alu transcription in vivo.

Cooperative Formation of Higher Order Peroxisome Proliferator-activated Receptor and Retinoid X Receptor Complexes on the Peroxisome Proliferator Responsive Element of the Rat Hydratase-Dehydrogenase Gene

Peroxisome proliferator-activated receptor (PPAR) forms a heterodimer with retinoid X receptor (RXR) that binds to the peroxisome proliferator responsive element (PPRE) to regulate the expression of target genes. PPRE of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) gene has previously been shown to consist of three imperfect TGACCT half-sites and form two distinct complexes (C1 and C2) with the nuclear extracts from H4IIEC3 cells. The present study identifies another imperfect TGACCT motif involved in the PPAR/RXR-mediated trans-activation process and demonstrates that these four imperfect TGACCT motifs constitute an unique binding site consisting of two DR1 elements overlapping a DR2 element. PPAR and RXR cooperatively bind the two DR1 elements to form C1 complex or bind DR2 element to form C2 complex with a 1:1 ratio. Saturation of the HD PPRE probes with receptor proteins cannot convert the heterodimeric C2 complex to the higher order C1 complex, suggesting that they are formed independently. Transfection analyses indicate that mutation of any one of these TGACCT motifs or truncation of the entire HD PPRE into a separate DR1 and DR2 element significantly reduced the transcriptional response of HD PPRE to peroxisome proliferators. The rat HD PPRE differentially binds with one or two PPAR/RXR heterodimers providing the peroxisome proliferator signaling pathway with two levels of response.