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PubMed1994

Cyclooxygenase and lipoxygenase inhibitors as modulators of cancer therapies.

Like many clinical non-small-cell lung cancers, the Lewis lung carcinoma produces prostaglandins. The Lewis lung carcinoma was used as a model of both primary and metastatic disease to assess the ability of cyclooxygenase inhibitors (mefenamic acid, diflunisal, sulindac, and indomethacin), the collagenase inhibitor minocycline, and the lipoxygenase inhibitor phenidone to act as modulators of cytotoxic cancer therapies. Although none of the single modulators given i.p. daily on days 4-18 altered tumor growth or the number of metastases found on day 20, modulator combinations consisting of minocycline/a cyclooxygenase inhibitor and, especially, of phenidone/a cyclooxygenase inhibitor resulted in modest tumor growth delay and a decreased number of lung metastases on day 20. The most effective modulators of cisplatin (CDDP) were phenidone/sulindac and phenidone/indomethacin, which led to 2.4- to 2.5-fold increases in the tumor growth delay produced by CDDP. The most effective modulations of cyclophosphamide resulted from administration of minocycline, minocycline/sulindac, or phenidone/sulindac and led to 2.0- to 2.1-fold increases in tumor growth delay by cyclophosphamide. The most effective modulators of melphalan produced 4.5- to 4.7-fold increases in tumor growth delay by the drug and were minocycline/sulindac, minocycline/mefenamic acid, and phenidone/sulindac. The most effective modulation of carmustine (BCNU) was obtained with minocycline/sulindac and minocycline/diflunisal leading to 2.8- to 3.1-fold increases in tumor growth delay by BCNU. Finally, the most effective modulation of radiation was obtained with minocycline/sulindac and phenidone/sulindac and resulted in 2.8- to 3.3-fold increases in tumor growth delay by radiation. The modulator combination that along with the cytotoxic therapies was most effective against metastatic disease was phenidone/mefenamic acid. There was no clear relationship between effective modulation of the cancer therapies and the degree of reduction in serum levels of prostaglandin E2 and leukotriene B4 by the agents in Lewis lung tumor bearing mice.

Animals↗
PubMed2004

Broad-spectrum modulation of ATP-binding cassette transport proteins by the taxane derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023.

PURPOSE: The taxanes paclitaxel and docetaxel are substrates for P-glycoprotein (Pgp), an ATP-binding cassette (ABC) transport protein associated with multidrug resistance (MDR). In contrast, the synthetic taxane ortataxel (BAY 59-8862, IDN-5109) is effective against Pgp-expressing cells by virtue of modulation of Pgp-mediated transport. The synthetic taxane tRA96023 also modulates Pgp and is noncytotoxic due to removal of the tubulin-binding side chain at the C-13 position of the taxane backbone. We studied the effects of ortataxel and tRA96023 on the other MDR-associated ABC transport proteins, multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP, MXR, ABCG2). METHODS: Modulation of mitoxantrone, daunorubicin and doxorubicin retention and cytotoxicity by ortataxel and tRA96023 was studied in established cell lines overexpressing Pgp, MRP-1 and wild type (BCRP(R482)) and mutant (BCRP(R482T)) BCRP, and was compared with modulation by the established Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C, respectively. RESULTS: Ortataxel effectively modulated drug retention and cytotoxicity in cell lines overexpressing MRP-1 and BCRP(R482), in addition to Pgp. tRA96023 modulated drug retention and cytotoxicity in cell lines overexpressing BCRP(R482) and Pgp, but not those overexpressing MRP-1. Neither ortataxel nor tRA96023 modulated BCRP(R482T). CONCLUSIONS: The synthetic taxane derivatives ortataxel and tRA96023 are broad-spectrum ABC protein modulators. Further studies will seek to identify a noncytotoxic synthetic taxane that modulates Pgp, MRP-1 and BCRP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Modulation and prevention of multidrug resistance by inhibitors of P-glycoprotein.

Intrinsic and acquired multidrug resistance (MDR) in many human cancers may be due to expression of the multidrug transporter P-glycoprotein (Pgp), which is encoded by the mdr1 gene. There is substantial evidence that Pgp is expressed both as an acquired mechanism (e.g., in leukemias, lymphomas, myeloma, and breast and ovarian carcinomas) and constitutively (e.g., in colorectal and renal cancers) and that its expression is of prognostic significance in many types of cancer. Clinical trials of MDR modulation are complicated by the presence of multiple-drug-resistance mechanisms in human cancers, the pharmacokinetic interactions that result from the inhibition of Pgp in normal tissues, and, until recently, the lack of potent and specific inhibitors of Pgp. A large number of clinical trials of reversal of MDR have been undertaken with drugs that are relatively weak inhibitors and produce limiting toxicities at doses below those necessary to inhibit Pgp significantly. The advent of newer drugs such as the cyclosporin PSC 833 (PSC) provides clinicians with more potent and specific inhibitors for MDR modulation trials. Understanding how modulators of Pgp such as PSC 833 affect the toxicity and pharmacokinetics of cytotoxic agents is fundamental for the design of therapeutic trials of MDR modulation. Our studies of combinations of high-dose cyclosporin (CsA) or PSC 833 with etoposide, doxorubicin, or paclitaxel have produced data regarding the role of Pgp in the clinical pharmacology of these agents. Major pharmacokinetic interactions result from the coadministration of CsA or PSC 833 with MDR-related anticancer agents (e.g., doxorubicin, daunorubicin, etoposide, paclitaxel, and vinblastine). These include increases in the plasma area under the curve and half-life and decreases in the clearance of these cytotoxic drugs, consistent with Pgp modulation at the biliary lumen and renal tubule, blocking excretion of drugs into the bile and urine. The biological and medical implications of our studies include the following. First, Pgp is a major organic cation transporter in tissues responsible for the excretion of xenobiotics (both drugs and toxins) by the biliary tract and proximal tubule of the kidney. Our clinical data are supported by recent studies in mdr-gene-knockout mice. Second, modulation of Pgp in tumors is likely to be accompanied by altered Pgp function in normal tissues, with pharmacokinetic interactions manifesting as inhibition of the disposition of MDR-related cytotoxins (which are transport substrates for Pgp). Third, these pharmacokinetic interactions of Pgp modulation are predictable if one defines the pharmacology of the modulating agent and the combination. The interactions lead to increased toxicities such as myelosuppression unless doses are modified to compensate for the altered disposition of MDR-related cytotoxins. Fourth, in serial studies where patients are their own controls and clinical resistance is established, remissions are observed when CsA or PSC 833 is added to therapy, even when doses of the cytotoxin are reduced by as much as 3-fold. This reversal of clinical drug resistance occurs particularly when the tumor cells express the mdr1 gene. Thus, tumor regression can be obtained without apparent increases in normal tissue toxicities. In parallel with these trials, we have recently demonstrated in the laboratory that PSC 833 decreases the mutation rate for resistance to doxorubicin and suppresses activation of mdr1 and the appearance of MDR mutants. These findings suggest that MDR modulation may delay the emergence of clinical drug resistance and support the concept of prevention of drug resistance in the earlier stages of disease and the utilization of time to progression as an important endpoint in clinical trials. Pivotal phase III trials to test these concepts with PSC 833 as an MDR modulator are under way or planned for patients with acute myeloid leukemias, multiple myeloma, and ovarian carcinoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Electron spin echo envelope modulation spectroscopy of the molybdenum center of xanthine oxidase.

The pulsed EPR technique of electron spin echo envelope modulation (ESEEM) has been utilized to examined both the 'very rapid' and 'desulfo inhibited' Mo(V) signals of xanthine oxidase in order to probe for magnetic interactions with nitrogen, phosphorus and hydrogen nuclei. No 14N modulation is observed in the 'desulfo inhibited' EPR signal, indicating that histidine is unlikely to be a ligand to molybdenum. Strong 14N modulation is observed in the 'very rapid' EPR signal formed with 2-hydroxy-6-methylpurine substrate bound to molybdenum. We interpret this modulation as arising from nitrogens of the bound purine substrate. This interpretation is consistent with the present evidence indicating that the purine ring present in the species giving rise to the 'very rapid' EPR signal is coordinated to the molybdenum center through the catalytically introduced hydroxyl group. No modulation is observed from non-exchangeable deuterons in experiments performed with deuterated 2-hydroxy-6-methylpurine. Given the signal-to-noise level of the spectra, the lack of modulation indicates that each of the substrate methyl group deuterons is greater than 4.9 A from the Mo(V). The deuteron removed from the C8 position in the binding of the substrate is also exchanged to a site or sites greater than 4.9 A from the Mo(V) in the time-course of sample preparation. Moderately deep deuteron modulation arises from exchangeable sites. A large portion of this modulation can be accounted for by the exchangeable N7 deuteron of the 2-hydroxy-6-methylpurine substrate, which we estimate to be approximately 3.2 A from the molybdenum. Additional exchangeable deuterons on the protein or within the buffer must be present within 5 A of the molybdenum to account for the remaining modulation. No modulation from weakly-coupled 31P nuclei is observed in either the 'desulfo inhibited' or 'very rapid' EPR signal.

Deuterium↗
PubMed1994

Identification of a multidrug resistance modulator with clinical potential by analysis of synergistic activity in vitro, toxicity in vivo and growth delay in a solid human tumour xenograft.

Circumvention of multidrug resistance in vitro by resistance modulators is well documented but their clinical use may be limited by effects on normal tissues. We have compared four resistance modifiers, both in terms of modulation of doxorubicin sensitivity in vitro and toxicity in vivo, in order to determine whether it is possible to select agents with clinical potential. Verapamil, D-verapamil and quinidine are all maximally active in the multidrug resistant cell line at about 7 microM and are not cytotoxic at this concentration. The tiapamil analogue Ro11-2933 is a highly potent resistance modulator such that at only 2 microM sensitization is greater than is seen with the other modulators at 7 microM. Since the ID50 concentration for Ro11-2933 is 17.7 microM (5-12-fold less than the other modifiers) we have used isobologram analysis to demonstrate that the interaction with doxorubicin is supra-additive and cannot be explained by additive toxicity. This method of analysis also revealed that when resistance modulation is related to the cytotoxicity of the modulator itself, all four modulators show comparable activity. On the other hand, measurement of the acute toxicity in mice of the modulators did reveal differences. The LD10 for verapamil (51 mg/kg) was about one third of that for quinidine (185 mg/kg) and this is consistent with the known maximum tolerated plasma levels in patients. Furthermore, whilst epirubicin alone was unable to reduce the growth rate of a multidrug resistant human tumour xenograft, the addition of quinidine, but not verapamil, at the maximum tolerated dose did do so. D-Verapamil was only about half as toxic as racemic verapamil and this too is consistent with clinical observations. The LD10 for Ro11-2933 (152 mg/kg) was comparable with that for quinidine. In the human tumour xenograft model maximal growth inhibition was observed with the combination of epirubicin and Ro11-2933 (45 mg/kg) and this degree of growth inhibition was comparable to that obtained with epirubicin alone in the drug sensitive xerografts. Ro11-2933 had no measurable effects on the plasma or tumour pharmacokinetics of epirubicin. These results suggest that it is possible to predict the clinical potential of a resistance modulator. Furthermore, Ro11-2933 is a promising agent for use in the clinic since maximal resistance modulation in vivo is observed at about one third of the LD10 dose.

Animals↗
PubMed1986

Sensitivity of neurons in the auditory midbrain of the grassfrog to temporal characteristics of sound. II. Stimulation with amplitude modulated sound.

The coding of fine-temporal structure of sound, especially of frequency of amplitude modulation, was investigated on the single-unit level in the auditory midbrain of the grassfrog. As stimuli sinusoidally amplitude modulated sound bursts and continuous sound with low-pass Gaussian noise amplitude modulation have been used. Both tonal and wideband noise carriers have been applied. The response to sinusoidally amplitude modulated sound bursts was studied in two aspects focussing on two types of possible codes: a rate code and a synchrony code. From the iso-intensity rate histogram five basic average response characteristics as function of modulation frequency have been observed: low-pass, band-pass, high-pass, bimodal and non-selective types. The synchronization capability, expressed in a synchronization index, was non-significant for 38% of the units and a low-pass function of modulation frequency for most of the other units. The stimulus-response relation to noise amplitude modulated sound was investigated by a non-linear system theoretical approach. On the basis of first- and second-order Wiener-Volterra kernels possible neural mechanisms accounting for temporal selectivity were obtained. About one quarter of the units had response characteristics that were invariant to changes in sound pressure level and spectral content of the carrier. These units may function as feature detectors of fine-temporal structure of sound. The spectro-temporal sensitivity range of the auditory midbrain of the grassfrog appeared not to be restricted to and showed no preference for the spectro-temporal characteristics of the ensemble of conspecific calls. Comparison of response characteristics to periodic click trains as studied in the companion paper (Epping and Eggermont, 1986) and sinusoidally amplitude modulated sound bursts revealed that the observed temporal sensitivity is due to a combination of sensitivities to sound periodicity and pulse duration. It was found that for most units the first-order kernels for Gaussian amplitude modulated stimuli and Poisson distributed click stimuli were alike. In contrast second-order kernels for the Gaussian amplitude modulated stimuli often represented only static non-linearities, while second-order kernels for Poisson distributed clicks (Epping and Eggermont, 1986) mostly revealed dynamic non-linearities.

Acoustic Stimulation↗
PubMed1993

Psychophysical frequency modulation thresholds in a FM-bat, Tadarida brasiliensis.

Echolocating bats hunting flying insects discriminate complex temporal patterns of acoustic stimuli. For bats using frequency modulated sonar calls (FM bats), there are no behavioral data on the perception of sinusoidally frequency modulated (SFM) stimuli. Discrimination performance for SFM stimuli of varying modulation depth was measured in 4 Tadarida brasiliensis in a two-alternative, forced choice procedure. A center frequency of 40 kHz was modulated with rates between 10 and 2000 Hz. It was found that discrimination performance improved from a mean threshold modulation depth of 3.05 kHz at a modulation rate of 2000 Hz to 1.58 kHz at a modulation rate of 10 Hz. Psychoacoustical modulation depth thresholds of T. brasiliensis are thus distinctly larger than those observed in bat species emitting constant frequency (CF) components followed by an FM-sweep, in active echolocation experiments. The modulation thresholds of T. brasiliensis are discussed in connection with the ability of bats to discriminate insect wingbeats. A comparison between non-echolocating mammals and the FM bat T. brasiliensis shows that the ability to echolocate is not reflected in the modulation thresholds.

Acoustic Stimulation↗
PubMed1994

Modulation detection interference: across-frequency processing and auditory grouping.

Modulation Detection Interference (MDI) is the loss of sensitivity in processing amplitude modulation of a probe tone when a masker is similarly modulated. MDI was measured in four experiments to investigate two past claims concerning MDI: 1) That MDI represents across-spectral processing, and 2) that MDI is the consequence of the auditory system using common patterns of amplitude modulation to group spectral components into a single auditory source. Experiment I studied MDI when the envelope phase of the masker and probe modulators were different and was used to address the issue of the extent to which MDI is a consequence of spectral grouping based on common amplitude modulation. Measures of MDI for conditions in which the frequency separation between the probe and masker carriers was varied (Experiment II), estimates of modulation depth discrimination (Experiment III), and signal detection thresholds for brief sinusoidal signals masked by amplitude modulated tones (Experiment IV) were all used to address issues related to across-spectral processing of amplitude modulation. The conclusions of these studies is that MDI is largely an across-frequency phenomenon and that the role of auditory grouping based on a common pattern of modulation can not be ruled out as having a relationship to MDI.

Acoustic Stimulation↗
PubMed2003

Temporal properties of the visual responses to luminance and contrast modulated noise.

Vision is sensitive to first-order luminance modulations and second-order modulations of carrier contrast. Our knowledge of the temporal properties of second-order vision is insufficient and contradictory. Using temporal summation and reaction time paradigms, we found that the type of visual noise (static or dynamic) determines the temporal properties of the responses to luminance and contrast modulations. In the presence of static noise, the temporal responses to both types of modulation of low and higher spatial frequencies were transient. When dynamic noise was used, the temporal responses to luminance and contrast modulations of higher spatial frequencies were sustained. At low spatial frequency, however, luminance modulations elicited transient responses, while contrast modulated dynamic noise produced sustained responses. The reaction times to near-threshold contrast modulations of low spatial frequency were slower than those to first-order patterns and they did not significantly differ at modulations of higher spatial frequency. The results suggest that the temporal characteristics of first-stage linear filters which feed the second-order pathway may determine the temporal responses to contrast modulated noise.

Contrast Sensitivity↗
PubMed2002

Hydrogen exchange, core modules, and new designed proteins.

A strategy for design of new proteins that mimic folding properties of native proteins is based on peptides modeled on the slow exchange cores of natural proteins. We have synthesized peptides, called core modules, that correspond to the elements of secondary structure that carry the very slowest exchanging amides in a protein. The expectation is that, if soluble in water, core modules will form conformational ensembles that favor native-like structure. Core modules modeled on natural bovine pancreatic trypsin inhibitor have been shown by NMR studies to meet this expectation. The next step toward production of a native state mimic is to further shift the conformational bias of a core module toward more ordered structure by promoting module-module interactions that are mutually stabilizing. For this, two core modules were incorporated into a single molecule by means of a long cross-link. From a panel of several two-module peptides, one very promising lead emerged; it is called BetaCore. BetaCore is monomeric in water and forms a new fold composed of a four-stranded, antiparallel beta-sheet. The single, dominant conformation of BetaCore is characterized by various NMR experiments. Here we compare the individual core module to the two-module BetaCore and discuss the progressive stabilization of intramodule structure and the formation of new intermodule interactions.

Amino Acid Sequence↗
PubMed1998

Potential clinical efficacy of intensity-modulated conformal therapy.

PURPOSE: The purpose of this study was to examine the potential benefit of using intensity-modulated conformal therapy for a variety of lesions currently treated with stereotactic radiosurgery or conventional radiotherapy. METHODS AND MATERIALS: Intensity-modulated conformal treatment plans were generated for small intracranial lesions, as well as head and neck, lung, breast, and prostate cases, using the Peacock Plan treatment-planning system (Nomos Corporation). For small intracranial lesions, intensity-modulated conformal treatment plans were compared with stereotactic radiosurgery treatment plans generated for patient treatment at the University of Florida Shands Cancer Center. For other sites (head and neck, lung, breast, and prostate), plans generated using the Peacock Plan were compared with conventional treatment plans, as well as beam's-eye-view conformal treatment plans. Plan comparisons were accomplished through conventional qualitative review of two-dimensional (2D) dose distributions in conjunction with quantitative techniques, such as dose-volume histograms, dosimetric statistics, normal tissue complication probabilities, tumor control probabilities, and objective numerical scoring. RESULTS: For small intracranial lesions, there is little difference between intensity-modulated conformal treatment planning and radiosurgery treatment planning in the conformation of high isodose lines with the target volume. However, stereotactic treatment planning provides a steeper dose gradient outside the target volume and, hence, a lower normal tissue toxicity index. For extracranial sites, objective numerical scores for beam's-eye-view and intensity-modulated conformal planning techniques are superior to scores for conventional treatment plans. The beam's-eye-view planning technique prevents geographic target misses and better excludes healthy tissues from the treatment portal. Compared with scores for the beam's-eye-view planning technique, scores for intensity-modulated conformal plans using the Peacock Plan were significantly better for the lung and head and neck cases studied, equivalent for prostate cases, and inferior for breast cases. CONCLUSION: Using the entire 3D data set to construct radiotherapy plans through virtual simulation is always advantageous, whether done for stereotactic radiosurgery, beam's-eye-view conformal therapy, or intensity-modulated conformal treatment. Intensity modulation of the photon beam further enhances treatment planning under specific conditions. In general, the intensity-modulated technique is advantageous for large, irregular targets with critical structures in close proximity. Intensity-modulated treatment planning does not appear advantageous for stereotactic radiosurgery or treatment of the intact breast.

Aged↗
PubMed1997

Temporal encoding and transmitting of amplitude and frequency modulations in dorsal cochlear nucleus.

Amplitude- and frequency-modulated (AM and FM, respectively) tones have been considered as simplified models of natural sounds. The responses of auditory neurons can phase-lock to the modulation frequency (fm). The encoding and transmitting of such modulation phase-locking are interesting since there is no any fm physical peak in spectrum. In the present study, we approached these issues by recording the phase-locked responses of the dorsal cochlear nucleus (DCN) units in guinea pigs to different AM and FM tones. For AM noise tones without the spectral cues of fm, the unit's discharges still phase-locked to the envelope cycles, but it was generally weaker than to sinusoidal AM (SAM) tones. At 50% modulation depth (dm), the mean modulation gains of Pauser/ Buildup (P/B) units (n = 7) to AM noise tones was -0.61 dB whereas they had a 6.48 dB mean to SAM tones. Similar to the case of AM tones, phase-locking to sinusoidal FM (SFM) tones represented the time courses of frequency changes, and it could be separated and changeable corresponding to the frequency increasing and decreasing. There were differences between the phase-locking to SAM and SFM tones in an identical unit. Both ON and type I/III units tended to have stronger phase-locking to the SFM tones than to the SAM tones. The phase-locking to the possible demodulated fm components was further examined with different carrier frequencies (fc) and pure tones. The DCN units showed poor or no responses to modulation tones out of their response areas even in the low characteristic frequency (CF) units, but the low-CF units had clear phase-locking to pure tones at the similar fm ranges. The puretone phase-locking had a band-pass shape different from the low-pass shape of the auditory nerve fibers. These data suggest that the modulation phase-locking in the DCN units may be based on the temporal modulation cues and transmitted in the carrier place. The temporal integration of modulation information over the unit's response area as an across-frequency temporal processing model was discussed for modulation enhancement in the CN units.

Acoustic Stimulation↗
PubMed1998

Processing of sinusoidally amplitude modulated signals in the nuclei of the lateral lemniscus of the big brown bat, Eptesicus fuscus.

Changes in amplitude are a characteristic feature of most natural sounds, including the biosonar signals used by bats for echolocation. Previous evidence suggests that the nuclei of the lateral lemniscus play an important role in processing timing information that is essential for target range determination in echolocation. Neurons that respond to unmodulated tones with a sustained discharge are found in the dorsal nucleus (DNLL), intermediate nucleus (INLL) and multipolar cell division of the ventral nucleus (VNLLm). These neurons provide a graded response over a broad dynamic range of intensities, and would be expected to provide information about the amplitude envelope of a modulated signal. Neurons that respond only at the onset of a tone make up a small proportion of cells in DNLL, INLL and VNLLm, but are the only type found in the columnar division of the ventral nucleus (VNLLc). Onset neurons in VNLLc maintain a constant latency across a wide range of stimulus frequencies and intensities, thus providing a precise marker for when a sound begins. To determine how these different functional classes of cells respond to amplitude changes, we presented sinusoidally amplitude modulated (SAM) signals monaurally to awake, restrained bats and recorded the responses of single neurons extracellularly. There were clear differences in the ability of neurons in the different cell groups to respond to SAM. In the VNLLm, INLL and DNLL, 90% of neurons responded to SAM with a synchronous discharge. Neurons in the VNLLc responded poorly or not at all to SAM signals. This finding was unexpected given the precise onset responses of VNLLc neurons to unmodulated tones and their ability to respond synchronously to sinusoidally frequency modulated (SFM) signals. Among neurons that responded synchronously to SAM, synchronization as a function of modulation rate described either a bandpass or a lowpass function, with the majority of bandpass functions in neurons that responded to unmodulated tones with a sustained discharge. The maximal modulation rates that elicited synchronous responses were similar for the different cell groups, ranging from 320 Hz in VNLLm to 230 Hz in DNLL. The range of best modulation rates was greater for SAM than for SFM; this was also true of the range of maximal modulation rates at which synchronous discharge occurred. There was little correlation between a neuron's best modulation rate or maximal modulation rate for SAM signals and those for SFM signals, suggesting that responsiveness to amplitude and frequency modulations depends on different neural processing mechanisms.

Acoustic Stimulation↗
PubMed2001

Solving modulated structures by X-ray and electron crystallography.

X-ray diffraction can be used for accurately determining not only classical, ordinary structures, but also modulated ones. For structures with weak modulations, the modulation induced satellite reflections are often hard to be observed by X-ray diffraction, but they appear clearly in electron diffraction. In these cases, X-ray diffraction will give only average structures whereas electron diffraction will yield information about the modulations. Sr(1.4)Ta(0.6)O(2.9) is a complex modulated compound with weak modulation and small modulated domains. Here we demonstrate the power of combining X-ray and electron crystallography for studying modulated structures on powders. The modulations of Sr(1.4)Ta(0.6)O(2.9) were determined from electron diffraction (SAED) and high resolution electron microscopy (HREM) images. With specially developed image processing techniques, the weak modulations were enhanced, facilitating the interpretation of HREM images in terms of atomic structure.

Journal Article↗
PubMed1999

Solution structure of a pair of modules from the gelatin-binding domain of fibronectin.

BACKGROUND: Fibronectin has a role in vital physiological processes such as cell migration during embryogenesis and wound healing. It mediates the attachment of cells to extracellular matrices that contain fibrous collagens. The affinity of fibronectin for native collagen and denatured collagen (gelatin) is located within a 42 kDa domain that contains four type 1 (F1) and two type 2 (F2) modules. A putative ligand-binding site has been located on an isolated F2 module, but the accessibility of this site in the intact domain is unknown. Thus, structural studies of module pairs and larger fragments are required for a better understanding of the interaction between fibronectin and collagen. RESULTS: The solution structure of the 101-residue 6F1 1F2 module pair, which has a weak affinity for gelatin, has been determined by multidimensional NMR spectroscopy. The tertiary structures determined for each module conform to the F1 and F2 consensus folds established previously. The experimental data suggest that the two modules interact via a small hydrophobic interface but may not be tightly associated. Near-random-coil 1H NMR chemical shifts and fast dynamics for backbone atoms in the linker indicate that this region is unlikely to be involved in the overall stabilisation of the module pair. CONCLUSIONS: The modules in the 6F1 1F2 module pair interact with each other via a flexible linker and a hydrophobic patch, which lies on the opposite side of the 1F2 module to the putative collagen-binding site. The intermodule interaction is relatively weak and transient.

Amino Acid Sequence↗
PubMed1997

Solution structure of a type 2 module from fibronectin: implications for the structure and function of the gelatin-binding domain.

BACKGROUND: Fibronectin is an extracellular matrix glycoprotein involved in cell adhesion and migration events in a range of important physiological processes. Aberrant adhesion of cells to the matrix may contribute to the breakdown of normal tissue function associated with various diseases. The adhesive properties of fibronectin may be mediated by its interaction with collagen, the most abundant extracellular matrix protein. The collagen-binding activity of fibronectin has been localized to a 42 kDa proteolytic fragment on the basis of this fragment's affinity for denatured collagen (gelatin). This gelatin-binding domain contains the only type 2 (F2) modules found in the protein. The F2 modules of the matrix metalloproteinases MMP2 and MMP9 are responsible for the affinity of these proteins for gelatin. Knowledge of the structure of fibronectin will provide insights into its interactions with other proteins, and will contribute to our understanding of the structure and function of the extracellular matrix, in both normal and disease-altered tissues. RESULTS: We have determined the solution structure of the first F2 (1F2) module from human fibronectin by two-dimensional NMR spectroscopy. The tertiary structure of the 1F2 module is similar to that of a shorter F2 module, PDC-109b, from the bovine seminal plasma protein PDC-109. The 1F2 module has two double-stranded antiparallel beta sheets oriented approximately perpendicular to each other, and enclosing a cluster of highly conserved aromatic residues, five of which form a solvent-exposed hydrophobic surface. The N-terminal extension in 1F2 brings the N and C termini of the module into close proximity. CONCLUSIONS: The close proximity of the N and C termini in 1F2 allows for interactions between non-contiguous modules in the gelatin-binding domain. Thus, instead of forming an extended, linear chain of modules, the domain may have a more compact, globular structure. A pocket in the module's solvent-exposed hydrophobic surface may bind nonpolar residues in the putative fibronectin-binding site of the extracellular matrix component type I collagen.

Amino Acid Sequence↗
PubMed2006

Modulation ratio in comprehensive two-dimensional gas chromatography.

Comprehensive two-dimensional chromatography employs a serially coupled two-column arrangement where effluent from the first column is collected or sampled and then introduced to the second column according to a chosen modulation period. This is effected by use of a modulator at or near the column junction. One of the considerations in applying the technique is the period of the modulator, which determines the sampling duration of the first column effluent. Here, we propose that the sampling rate can be most effectively described by a new term, called the modulation ratio (MR). This is defined as the ratio of 4 times the first column peak standard deviation (4sigma) divided by the modulation period (PM) or 1.6985 times the half-height width of the peak (wh): MR = 4sigma/PM = wb/PM = (wh x 1.6985)/PM. The 4sigma value is more commonly recognized as the peak base width (wb). The use of 4sigma as the numerator is preferred to simply sigma because when the PM value used for an experiment is equal to sigma, then the MR value is calculated to be 4, implying that the primary peak will be modulated approximately 4 times as is normally recommended for a comprehensive multidimensional separation. The less well-defined term of modulation number (NM) has been previously used and proposed as the number of modulations per peak and, therefore, is intended to convey the manner in which the primary column peak is sampled; this is a subjective and not well-characterized value. The use of MR should provide users with a meaningful and strictly defined value when reporting experimental conditions. The utility of MR is demonstrated through a mathematical model of the modulation process for both Gaussian and tailing peaks, supported by an experimental study of the modulation ratio. It is shown that for the analysis of trace compounds where precise quantitative measurements are being made, the experiment should be conducted with an MR of at least 3. Conversely, for semiquantitative methods or the analysis of major components, an MR of approximately 1.5 should suffice.

Journal Article↗
PubMed2004

Intrasteric inhibition mediates the interaction of the I/LWEQ module proteins Talin1, Talin2, Hip1, and Hip12 with actin.

The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. Previous studies have shown that I/LWEQ module proteins bind to F-actin. In this report, we have determined the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. Our data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. We have also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis.

Actins↗