Eustachian valve endocarditis: Detection with multiplane transesophageal echocardiography

Right-sided involvement is fairly common in infective endocarditis, but involvement of the eustachian valve is distinctly rare. We present the case of a 36-year-old intravenous drug user with staphylococcal bacteremia and septic pulmonary emboli. Transthoracic echocardiography was normal, but transesophageal echocardiography revealed a large eustachian valve vegetation. This case illustrates the utility of multiplane transesophageal echocardiography in the evaluation of eustachian valve pathology.     

Pediatric interstitial lung disease revisited

The spectrum of pediatric interstitial lung disease (PILD) includes a diverse group of rare disorders characterized by diffuse infiltrates and disordered gas exchange. Children with these conditions typically present with tachypnea, crackles, and hypoxemia. Recent advances have been made in the identification of different types of PILD that are unique to infancy. More exciting has been the discovery of genetic abnormalities of surfactant function, now described in both children and adults. A systematic evaluation of the child presenting with diffuse infiltrates of unknown etiology is essential to the diagnosis. Most often, lung biopsy is required. Current treatment options remain less than satisfactory, and morbidity and mortality remain considerable.     

Discordance of exercise thallium testing with coronary arteriography in patients with atypical presentations

Eighty-one patients with diagnostically difficult clinical presentations suggesting coronary disease underwent symptom-limited maximal-exercise treadmill testing (ETT) and exercise radionuclide scanning with thallium-201. Results of these tests were in agreement in only 47 percent of the cases. Either exercise thallium or ETT was positive in 94 percent of patients with disease. Among a population with a disease prevalence of 67 percent, agreement between exercise thallium an ETT predicted disease in 92 percent of instances or excluded disease in 82 percent of instances. Frequent discordance between these two tests in 53 percent of the cases unfortunately limits this usefulness.     

Protein hydrogen exchange at residue resolution by proteolytic fragmentation mass spectrometry analysis

Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange–mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved back-exchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.Unlike any other method, hydrogen exchange (HX) behavior encodes detailed quantitative information at amino acid resolution on the biophysical factors that produce protein function—structure, structure change, interactions, dynamics, and energetics. HX behavior is very sensitive to these properties, and the chemistry (1, 2) and structural physics (3, 4) of HX processes in these terms are now well understood. The ability of HX to measure protein biophysical properties and their implementation in protein function has been demonstrated over the last 30 y by many NMR studies. However, routine NMR analysis is limited to small highly soluble proteins that can be obtained in quantity (multi mgs), isotopically labeled (¹⁵N, ¹³C), and studied at millimolar concentration. A developing technology, HX measured and analyzed by mass spectrometry (HX MS) (5–16) can extend this proven capability to the much larger and more complex protein systems that make biology work. The method requires only picomoles of protein at submicromolar concentrations; it can be used to study the properties and functioning of proteins at any condition one chooses, and the experimental protein need not even be very pure.For HX MS analysis, a protein sample taken from any H–D exchange experiment is quenched into slow HX conditions, proteolytically fragmented, and the peptide fragments are separated and analyzed by HPLC and mass spectrometry. The number of D atoms carried on each peptide fragment is given by its measurable increase in mass, usually calculated as the increment in the peptide mass centroid. These results provide structural information resolved to the level of individual fragments and are broadly able to indicate where in the protein important behavior occurs (5–16). More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Recently reported efforts have moved in this direction (15, 17–24).We find that the effort to achieve residue resolution is not limited by a difficult calculational barrier (17, 18, 20) but by experimental difficulties. To reach this goal, it is necessary to obtain many sequentially overlapping peptide fragments that cover the experimental protein several times over. It is also necessary to solve the back-exchange problem, the loss of D label, and the information it carries, which unavoidably occurs during the sample preparation process before sample injection into the mass spectrometer. We have described experimental and computational methods that can produce and rigorously identify and characterize hundreds of peptide fragments (25, 26) and that minimize back exchange (27). This paper demonstrates a straightforward approach that uses these capabilities together with previously unexamined envelope-shape information (isotopic peak amplitude ratios) and a site-resolved back-exchange correction to analyze HX MS results to amino acid resolution.     

Folding of a large protein at high structural resolution

Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.Fifty years after Anfinsen’s seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3–7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8–12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work has demonstrated an advanced hydrogen-exchange pulse-labeling mass-spectrometry technology (HX MS) that is able to detect and characterize local structure, even when it is only transiently present during the course of kinetic folding (15, 16). The HX pulse-labeling approach provides a snapshot of main chain amide sites that are protected against HX labeling by H bonds present at the time of the labeling pulse (17, 18). HX MS measurements can determine the position, stability, and dynamic behavior of native and nonnative H-bonded structure and whether it persists or dissipates in subsequent folding. In a recent application, the method was able to describe the structure and time-dependent formation of three sequential native-like folding intermediates in the 155-residue ribonuclease H protein (15).Protein folding studies, whether theoretical or experimental, have been limited to relatively small proteins, with few exceptions. However, biological proteomes and the considerations they raise are dominated by large proteins (19). Here we extend the powerful HX MS technology to the two-domain, 370-residue, maltose binding protein (MBP). MBP is synthesized in the Escherichia coli cytoplasm and transported to the periplasm where it serves as a soluble receptor for the high-affinity capture and import of maltose and maltodextrins (20). The protein folds in vivo after deletion of a signal sequence (21); we study here the mature protein with the signal sequence deleted.When unfolded MBP is placed into native conditions, we find that it rapidly adopts a dynamic collapsed state, which can lead to aggregation in vitro when the concentration is >1 μM and to inclusion body formation in vivo (22). Folding to the native state occurs much more slowly even in the absence of aggregation, with all molecules moving through one or more intermediate states to the native state. The HX MS experiment provides incisive information on the nature of the initially collapsed state, the slow formation and identity of at least one on-pathway native-like intermediate, and the even slower emergence of native structure.     

Expression of high-affinity IL-4 receptors on murine tumour infiltrating lymphocytes and their up-regulation by IL-2.

Since interleukin-2 (IL-2) and IL-4 act in concert to support the development of cytotoxic T lymphocytes (CTL) and the generation of antigen-specific tumour infiltrating lymphocytes (TIL), we investigated the interaction of these cytokines with an established TIL line. TIL proliferated in an additive fashion in response to suboptimal concentrations of IL-2 and various concentrations of IL-4. TIL possessed high-affinity IL-4 receptors whether cultured in recombinant IL-2 (rIL-2) or rIL-4, but cells cultured in rIL-2 had higher numbers of IL-4 receptors than cells cultured in rIL-4. When TIL were cultured in increasing concentrations of rIL-2, a dose-dependent enhancement in IL-4 receptor number was observed. The maximum induction of IL-4 receptor expression was achieved by 4 hr of incubation with rIL-2 and was completely blocked by cycloheximide. Other cytokines, such as rIL-1, recombinant tumour necrosis factor (rTNF), recombinant interferon-alpha (rIFN-alpha) and rIFN-gamma, had no effect on IL-4 receptor number. rIL-2 also up-regulated IL-4 receptors on CTLL-2, a murine CTL line. These data indicate that high-affinity IL-4 receptors exist on murine TIL and they can be up-regulated by IL-2. Our observation that IL-2 up-regulates IL-4 receptor may help explain the additive effects of these lymphokines on the proliferation of TIL and other cell lines. It may also help explain their co-operative effects on the generation of antigen-specific TIL and the differentiation of CTL.     

Treatment of general paralysis by radiothermy

Summary The clinical and laboratory results of 68 patients with general paralysis treated by radiothermy are compared with a similar number of patients with general paralysis treated by malaria. The two groups were as similarly constituted as could reasonably be expected.Improvement from the clinical standpoint was found to be about the same in each series of 68 patients. It was in the neighborhood of 53 per cent for each group. The clinical remission, percentage was 17.6 in the group treated by radiothermy as compared with 19.1 in the malarial-treated group. The relatively large proportion of women in these related groups seems to account for the comparatively low remission rates.The results from the laboratory standpoint are quite evidently inferior to those commonly observed in malarial-treated patients.From the Clinical Department of the New York State Psychiatric Institute and Hospital, New York, N. Y.