Defective plasmacytoid dendritic cell-NK cell cross-talk in HIV infection.

HIV viremia is associated with a wide range of immune dysfunctions that contribute to the immunocompromised state. HIV viremia has been shown to have a broad effect on several immune cell types and/or their interactions that are vital for mounting an effective immune response. In this study, we investigated the integrity of plasmacytoid dendritic cell (pDC)-NK cell interactions among HIV viremic, aviremic, and seronegative individuals. We describe a critical defect in the ability of pDCs from HIV-infected individuals to secrete IFN-alpha and TNF and subsequently activate NK cells. We also describe an inherent defect on NK cells from HIV-infected individuals to respond to pDC-secreted cytokines. Furthermore, we were able to demonstrate a direct effect of HIV trimeric gp120 on NK cells in vitro similar to that described ex vivo. Finally, we were able to establish that the HIV gp120-mediated suppressive effect on NK cells was a result of its binding to the integrin alpha(4)beta(7) expressed on NK cells. These findings suggest a novel mechanism by which HIV is capable of suppressing an innate immune function in infected individuals.

World-wide radiation dosage calculations for air crew members.

A greatly improved version of the computer program to calculate radiation dosage to air crew members is now available. Designated CARI-6, this program incorporates an updated geomagnetic cutoff rigidity model and a revision of the primary cosmic ray spectrum based on recent work by Gaisser and Stanev (1998). We believe CARI-6 provides the most accurate available method for calculating the radiation dosage to air crew members. The program is now utilized by airline companies around the world and provides unification for subsequent world-wide studies on the effects of natural radiation on aircrew members.

Evaluation of the dynamic cutoff rigidity model using dosimetry data from the STS-28 flight.

We have a developed a dynamic cutoff rigidity model based on computed world grids of vertical cutoff rigidities derived from employing the Tsyganenko magnetospheric model. The dynamic range of this model covers all magnetic activity levels specified by integer values of the Kp magnetic index. We present comparisons of the measured dose observed on the space shuttle during the August 1989 solar proton event with the dose computed from solar particles predicted to be allowed through the magnetosphere to the space shuttle position. We find a one-to-one correspondence between the portion of the orbit predicted to be subjected to solar protons and the portion of the orbit where solar particle dose measurements were obtained.

Comment on estimating the solar proton environment that may affect Mars missions.

Estimates of the energetic proton environment for a Mars mission are generally extrapolated from the solar proton observations at 1 AU. We find that solar particle events may be divided into two general classes. Events dominated by a near-sun injection of particles onto interplanetary magnetic field lines leading to the spacecraft position represent the "classical" solar particle event associated with solar activity. This class of event will scale in radial distance by the classical power law extrapolation. The extended-interplanetary-shock source generates a maximum flux as the shock passes the detection location. This class of event typically generates maximum fluence, but in this case, the flux and fluence will not scale in the classical manner with radial distance.

Calculations and observations of solar particle enhancements to the radiation environment at aircraft altitudes.

Solar particle events can give greatly enhanced radiation at aircraft altitudes, but are both difficult to predict and to calculate retrospectively. This enhanced radiation can give significant dose to aircrew and greatly increase the rate of single event effects in avionics. Validation of calculations is required but only very few events have been measured in flight. The CREAM detector on Concorde detected the event of 29 September 1989 and also four periods of enhancement during the events of 19-24 October 1989. Instantaneous rates were enhanced by up to a factor ten compared with quiet-time cosmic rays, while flight-averages were enhanced by up to a factor six. Calculations are described for increases in radiation at aircraft altitudes using solar particle spectra in conjunction with Monte Carlo radiation transport codes. In order to obtain solar particle spectra with sufficient accuracy over the required energy range it is necessary to combine space data with measurements from a wide range of geomagnetically dispersed, ground-level neutron monitors. Such spectra have been obtained for 29 September 1989 and 24 October 1989 and these are used to calculate enhancements that are compared with the data from CREAM on Concorde. The effect of cut-off rigidity suppression by geomagnetic activity is shown to be significant. For the largest event on record on 23 February 1956, there are no space data but there are data from a number of ground-level cosmic-ray detectors. Predictions for all events show very steep dependencies on both latitude and altitude. At high latitude and altitude (17 km) calculated increases with respect to cosmic rays are a factor 70 and 500 respectively for 29 September 1989 and 23 February 1956. The levels of radiation for high latitude, subsonic routes are calculated, using London to Los Angeles as an example, and can exceed 1 mSv, which is significantly higher than for Concorde routes from Europe to New York. The sensitivity of the calculations to spectral fitting, geomagnetic activity and other assumptions demonstrates the requirement for widespread carriage of radiation monitors on aircraft.

The limitations of using vertical cutoff rigidities determined from the IGRF magnetic field models for computing aircraft radiation dose.

Vertical cutoff rigidities derived from the International Geomagnetic Reference Fields (IGRF) are normally used to compute the radiation dose at a specific location and to organize the radiation dose measurements acquired at aircraft altitudes. This paper presents some of the usually ignored limits on the accuracy of the vertical cutoff rigidity models and describes some of the computational artifacts present in these models. It is noted that recent aircraft surveys of the radiation dose experienced along specific flight paths is sufficiently precise that the secular variation of the geomagnetic field is observable.

The space-developed dynamic vertical cutoff rigidity model and its applicability to aircraft radiation dose.

We have developed a dynamic geomagnetic vertical cutoff rigidity model that predicts the energetic charged particle transmission through the magnetosphere. Initially developed for space applications, we demonstrate the applicability of this library of cutoff rigidity models for computing aircraft radiation dose. The world grids of vertical cutoff rigidities were obtained by particle trajectory tracing in a magnetospheric model. This reference set of world grids of vertical cutoff rigidities calculated for satellite altitudes covers all magnetic activity levels from super quiet to extremely disturbed (i.e., Kp indices ranging from 0 to 9+) for every three hours in universal time. We utilize the McIlwain "L" parameter as the basis of the interpolation technique to reduce these initial satellite altitude vertical cutoff rigidities to cutoff rigidity values at aircraft altitudes.

The local time dependence of the anisotropic solar cosmic ray flux.

The distribution of the solar cosmic radiation flux over the earth is not uniform, but the result of complex phenomena involving the interplanetary magnetic field, the geomagnetic field and latitude and longitude of locations on the earth. The latitude effect relates to the geomagnetic shield; the longitude effect relates to local time. For anisotropic solar cosmic ray events the maximum particle flux is always along the interplanetary magnetic field direction, sometimes called the Archimedean spiral path from the sun to the earth. During anisotropic solar cosmic ray event, the locations on the earth viewing "sunward" into the interplanetary magnetic field direction will observe the largest flux (when adjustments are made for the magnetic latitude effect). To relate this phenomena to aircraft routes, for anisotropic solar cosmic ray events that occur during "normal quiescent" conditions, the maximum solar cosmic ray flux (and corresponding solar particle radiation dose) will be observed in the dawn quadrant, ideally at about 06 hours local time.

A review of solar proton events during the 22nd solar cycle.

Solar cycle 22 had significant, large fluence, energetic particle events on a scale reminiscent of the 19th solar cycle. Examination of the characteristics of these large events suggests that some of the old concepts of spectral form, intensity-time envelope and energy extrapolations, used to estimate the dose from large events that occurred during previous solar cycles should be re-evaluated. There has also been a dramatic change in perspective regarding the source of solar protons observed in interplanetary space. Very large fluence events are associated with powerful fast interplanetary shocks. The elemental composition and charge state of these events is suggestive of a dominate source in the solar corona and not from a very hot plasma. Furthermore, there is a strong suggestion that the intensity-time profile observed in space is dominated by the connection of the observer to an interplanetary shock source rather than to a unique location near the surface of the sun. These concepts will be examined from the perspective of energetic particles contributing to the dose experienced by an astronaut on an interplanetary space mission.

The cosmic ray ground level enhancement of 6 November 1997.

The relativistic solar proton event of 6 November 1997 resulted in the first ground-level enhancement (GLE) of solar cycle 23. The earliest onset was around 1215 UT but was up to 15 minutes later at some neutron monitor locations. The time of maximum intensity also varied significantly over the world-wide neutron monitor network. The modeled particle distributions and spectra are presented. The apparent particle arrival direction is found to be largely consistent with propagation outward from the sun along interplanetary magnetic field lines.

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin.

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.

Phenylalanine fluorescence studies of calcium binding to N-domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder.

Calmodulin (CaM) is a ubiquitous, essential calcium-binding protein that regulates diverse protein targets in response to physiological calcium fluctuations. Most high-resolution structures of CaM-target complexes indicate that the two homologous domains of CaM are equivalent partners in target recognition. However, mutations between calcium-binding sites I and II in the N-domain of Paramecium calmodulin (PCaM) selectively affect calcium-dependent sodium currents. To understand these domain-specific effects, N-domain fragments (PCaM(1-75)) of six of these mutants were examined to determine whether energetics of calcium binding to sites I and II or conformational properties had been perturbed. These PCaM((1-75)) sequences naturally contain 5 Phe residues but no Tyr or Trp; calcium binding was monitored by observing the reduction in intrinsic phenylalanine fluorescence at 280 nm. To assess mutation-induced conformational changes, thermal denaturation of the apo PCaM((1-75)) sequences, and calcium-dependent changes in Stokes radii were determined. The free energy of calcium binding to each mutant was within 1 kcal/mole of the value for wild type and calcium reduced the R(s) of all of them. A striking trend was observed whereby mutants showing an increase in calcium affinity and R(s) had a concomitant decrease in thermal stability (by as much as 18 degrees C). Thus, mutations between the binding sites that increased disorder and reduced tertiary constraints in the apo state promoted calcium coordination. This finding underscores the complexity of the linkage between calcium binding and conformational change and the difficulty in predicting mutational effects.

Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse.

Calmodulin (CaM) is a small acidic protein essential to calcium-mediated signal transduction. Conformational change driven by calcium binding controls its selective activation of myriad target proteins. In most well characterized cases, both homologous domains of CaM interact with a target protein. However, physiologically separable roles for the two domains were demonstrated by mutants of Paramecium tetraurelia [Kung, C. et al. (1992) Cell Calcium 13, 413], some of which have altered calcium affinities [Jaren, O. R. et al. (2000) Biochemistry 39, 6881]. To determine whether these mutants can associate with canonical targets in a calcium-dependent manner, their ability to bind melittin was assessed using analytical gel permeation chromatography, analytical ultracentrifugation, and fluorescence spectroscopy. The Stokes radius of wild-type PCaM and 11 of the mutants decreased dramatically upon binding melittin in the presence of calcium. Fluorescence spectra and sedimentation velocity studies showed that melittin bound to wild-type PCaM and mutants in a calcium-independent manner. However, there were domain-specific perturbations. Mutations in the N-domain of PCaM did not affect the spectrum of melittin (residue W19) under apo or calcium-saturated conditions, whereas most of the mutations in the C-domain did. These data are consistent with a calcium-dependent model of sequential target association whereby melittin (i) binds to the C-domain of PCaM in the absence of calcium, (ii) remains associated with the C-domain upon calcium binding to sites III and IV, and (iii) subsequently binds to the N-domain upon calcium binding to sites I and II of CaM, causing tertiary collapse.

Proteolytic footprinting titrations for estimating ligand-binding constants and detecting pathways of conformational switching of calmodulin.

To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized "silent" interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibirum titrations monitored by NMR. By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one "direction" for comparison. Quantitative proteolytic footprinting titrations applied to studying calmodulin provided the first direct quantitative estimate of negative interactions between domains. Although studies of site-knockout mutants had suggested interactions between domains, this approach gave the first evidence for the pathway of anticooperative interactions between domains by showing that helix B responds structurally to calcium binding to sites III and IV in the C-domain. Despite two decades of study of calmodulin and the application of limited proteolysis studies to the apo and fully saturated forms, this finding emerged only when titration studies were undertaken as described. This highlights the general observation that while the behavior of the intermediate states in a cooperative switch are the key elements of the transition mechanism, they are the most difficult to observe. The unexpected finding that the isolated domains are nearly equivalent in their calcium-binding properties (Fig. 23 B) leaves us with many of the questions we had at the start: How does the sum of two nearly equivalent domains result in a molecule that switches sequentially rather than simultaneously? But it underscores why it is not yet possible to understand similar proteins by sequence gazing alone.

Paramecium calmodulin mutants defective in ion channel regulation can bind calcium and undergo calcium-induced conformational switching.

Calmodulin (CaM) is an essential eukaryotic protein that binds calcium ions cooperatively at four EF-hand binding sites to regulate signal transduction pathways. Interactions between the apo domains of vertebrate CaM reduce the calcium affinities of sites I and II below their intrinsic values, allowing sequential opening of the two hydrophobic clefts in CaM. Viable domain-specific mutants of Parameciumcalmodulin (PCaM) differentially affect ion channels and provide a unique opportunity to dissect the roles of the two highly homologous half-molecule domains. Calcium binding induced an increase in the level of ordered secondary structure and a decrease in Stokes radius in these mutants; such changes were identical in direction to those of wild type CaM, but the magnitude depended on the mutation. Calcium titrations monitored by changes in the intrinsic fluorescence of Y138 in site IV showed that the affinities of sites III and IV of wild type PCaM were (i) higher than those of the same sites in rat CaM, (ii) equivalent to those of the same sites in PCaM mutants altered between sites I and II, and (iii) higher than those of PCaM mutants modified in sites III and IV. Thus, calcium saturation drove all mutants to undergo conformational switching in the same direction but not to the same extent as wild type PCaM. The disruption of the allosteric mechanism that is manifest as faulty channel regulation may be explained by altered properties of switching among the 14 possible partially saturated species of PCaM rather than by an inability to adopt two end-state conformations or target interactions similar to those of the wild type protein.

Identification of major proton fluence events from nitrates in polar ice cores.

Large transient concentrations of nitrates in polar ice cores have been identified as the signature of some major solar proton fluence events between 1940 and 1991. We review this solar proton proxy identification technique using nitrate concentration measurements in ice cores from the Arctic and Antarctic. Using this identification technique we go back in time in an attempt to identify major solar proton events during the past several centuries. There is a very large nitrate increase corresponding to the Carrington flare of 1859 evident in the Arctic ice core. Other significant nitrate increases may indicate that major solar proton events occurred toward the end of the last century. The problems associated with this new technique of using nitrates as proxies to identify solar proton events are discussed.

Comment on the use of GOES solar proton data and spectra in solar proton dose calculations.

There is a need to understand the calibration and response of the GOES solar particle detectors since the GOES data are being used to evaluate high energy solar particle events. We share some of our experience in utilizing these data in the analysis of solar particle ground-level events (GLEs). For the 29 September 1989 event, we have evaluated the solar proton and alpha particle spectral characteristics throughout the event. The results show that the solar cosmic ray spectrum is extremely hard at low energies with the magnitude of the slope increasing with increasing energy and with time.

Neoadjuvant chemoradiotherapy for esophageal cancer: is it worthwhile?

BACKGROUND: With promising results from several institutions, many centers began treating patients with esophageal cancer with neoadjuvant chemoradiotherapy (NC) followed by esophagectomy. This approach is demanding for the patient and has not been proved to be better than esophagectomy alone. OBJECTIVE: To assess survival time and measures of quality of life associated with NC. DESIGN: A retrospective review during 1990 to 1996. SETTING: The 3 tertiary academic hospitals affiliated with the University of Massachusetts Medical School, Worcester. PARTICIPANTS: All patients (N=51) with cancer of the middle or lower esophagus who were treated with NC followed by esophagectomy during this period. MAIN OUTCOME MEASURES: Median and 1-, 2-, and 3-year survival times; median preoperative treatment time (first office visit for surgical consultation before beginning NC to the date of surgery), median hospital stay, and postoperative swallowing function. RESULTS: The median survival time of all patients was 16.3 months; 1-, 2-, and 3-year overall survival rates were 67%, 46%, and 39%, respectively. The median hospital stay was 12 days. The median postoperative treatment time was 3.3 months, which was 20% of the median survival time. Of the 51 patients, 19 were alive with a median follow-up time of 2.5 years. Twenty-nine percent of the patients had a complete pathological response with median and 1-, 2-, and 3-year survival rates of 17.5 months, 73%, 57%, and 57%, respectively. Palliation of dysphagia was excellent, with 44 (93%) of 47 operative survivors taking either a soft diet (18 [38%]) or a regular (26 [55%]) diet by the first postoperative visit. CONCLUSIONS: Median survival time with NC followed by esophagectomy for resectable cancer of the esophagus does not appear to be significantly better than that reported for esophagectomy alone. Further, treatment time with NC consumed 20% of survival time. Examining only these outcome variables suggests that NC is not worth-while. However, examining a longer-term outcome survival variable, such as 3-year survival time, suggests that NC followed by esophagectomy may result in greater long-term survival than that reported for esophagectomy alone. We conclude that further randomized, controlled studies are necessary before NC followed by esophagectomy is considered superior to esophagectomy alone for the treatment of resectable esophageal cancer.

Interactions between domains of apo calmodulin alter calcium binding and stability.

Calmodulin (CaM) is an essential protein that exerts exquisite spatial and temporal control over diverse eukaryotic processes. Although the two half-molecule domains of CaM each have two EF-hands and bind two calcium ions cooperatively, they have distinct roles in activation of some targets. Interdomain interactions may mediate coordination of their actions. Proteolytic footprinting titrations of CaM [Pedigo and Shea (1995) Biochemistry 34, 1179-1196; Shea, Verhoeven, and Pedigo (1996) Biochemistry 35, 2943-2957] showed that calcium binding to the high-affinity sites (III and IV in the C-domain) alters the conformation of helix B in the N-domain despite sites I and II being vacant. This may arise from calcium-induced disruption of interactions between the apo domains. In this study, comparing the cloned domains (residues 1-75, 76-148) to whole CaM, the proteolytic susceptibility of helix B in the apo isolated N-domain was higher than in apo CaM. The isolated N-domain was monotonically protected by calcium binding and had a higher calcium affinity than when part of whole CaM. The change in affinity was small (1-1.5 kcal/mol) but acted to separate the domain saturation curves of whole CaM. Unfolding enthalpies and melting temperatures of the apo isolated domains did not correspond to the two transitions resolved for apo CaM. In summary, the interactions between domains of apo CaM protected the N-domain from proteolysis and raised its Tm by 10 degrees C, demonstrating that CaM is not the sum of its parts.

Expandable metallic esophageal stents in benign disease: a cause for concern.

Over a 2-year period at our institution, 6 patients underwent metallic stent treatment, 5 for malignant conditions and 1 for a benign condition of the esophagus. The use of expandable metallic stents for benign strictures has paralleled malignant indications but is limited and less understood from a clinical standpoint. A review of current literature in the treatment of benign strictures is presented. Treatment of benign strictures is associated with high morbidity and mortality as demonstrated by the cumulative experience of 21 patients. Migration, hyperplastic tissue obstruction at the terminal ends, reflux, and complications of perforation occur at a prohibitive rate. We conclude that expandable metallic stents should be reserved for palliative treatment of esophageal malignant obstructions and tracheoesophageal fistulas. Pharmacological management, necessary dilatations and operative corrections (antireflux procedures, esophagectomy) are recommended treatments for benign strictures.