Temsirolimus as base immunosuppressant for a recipient with metastatic renal cancer: adequate immunosuppression and oncological control--case report.

Treating organ recipients who have metastatic renal cell carcinoma (mRCC) is challenging because of the dilemma between transplant-required immunosuppression and cancer control via boosting immunity with immunotherapy. We report such a patient whose case was successfully maintained only with temsirolimus and low-dose steroids, while achieving good allograft function and oncological outcomes. After removal of his primary renal cancer, a kidney recipient was found to have multiple metastases. Since recovery from surgery he has been administered 25 mg/wk temsirolimus for 2 more years. His mRCC is in partial remission, the serum creatinine level has been stable (∼ 1.6 mg/dL), and the performance score has been good. Adverse effects encountered include transient transaminitis, dyslipidemia, and poorly controlled hyperglycemia. The pharmacokinetics of sirolimus (the major first metabolite of temsirolimus) is depicted to speculate the underlying mechanism. To our knowledge, this is the first sustainable success using temsirolimus-based immunotherapy (both immunosuppression and cancer therapy) in a transplant recipient who has mRCC.

Incorporation of single-walled carbon nanotubes into functional sensor applications.

Due to their extreme sensitivity to surface adsorption events, there has been great interest in incorporating single walled carbon nanotubes (SWNTs) into sensing devices. However, a practical sensor needs to have selective response in addition to sensitivity. Therefore, recent efforts have focused on efforts to increase selectivity in SWNT sensors. This manuscript article presents recent progress toward incorporation of SWNTs into enhanced sensor and electronic applications. SWNTs are a nanometer-scale material with properties that have made them the focus of research for a variety of electronic applications. Particularly, SWNT gas sensors have received significant interest of late. Electrical transducers based on monitoring the change in conductance or threshold voltage of SWNT devices upon exposure to an analyte have the advantage of being small, simple and low power devices.

The xenograft antigen in complex with GS-1-B4 lectin: crystallization and preliminary X-ray analysis.

The implantation of animal organs is one approach to overcoming the shortage of human donor organs for medical transplantation. Although readily available, non-primate tissues are subject to hyperacute rejection wherein human anti-Galalpha(1-3)Gal antibodies react with haptens present on the transplanted cells' surfaces. The understanding of this interaction on a molecular level will further the development of a strategy for the prevention of hyperacute rejection in xenotransplantation. The Galalpha(1-3)Gal hapten ('xenograft antigen') has been cocrystallized with the Gal-specific B(4) isolectin of Griffonia simplicifolia lectin-1. Crystals were analyzed by cryocrystallography and were found to diffract to moderately high resolution on a rotating-anode X-ray source. They belong to the P2(1)2(1)2 space group, with unit-cell parameters a = 111.0, b = 51.3, c = 76.9 A, and contain two molecules per asymmetric unit.

Medically recommended cessation of employment among pregnant women in Georgia.

OBJECTIVE: To ascertain the proportion of employed pregnant women who receive medical advice to stop working during pregnancy and to describe their characteristics. METHODS: Data were analyzed from the Georgia Pregnancy Risk Assessment Monitoring System, a surveillance system that surveys new mothers about pregnancy risk factors, health behaviors, and birth-related outcomes. Employment during pregnancy was defined as work for pay for 10 hours or more per week. RESULTS: We studied 1635 women who were employed during pregnancy. A physician or nurse had advised 27.7% (95% CI 24.5%, 30.9%) of them to stop working during pregnancy. Independent predictors of receiving this advice were hospitalization (RR 2.3, 95% CI 1.7, 2.8) and history of previous preterm birth (RR 1.6, 95% CI 1.1, 2.2). Low birth weight (under 2500 g) occurred in 5.8% of women not advised to stop work, in 6.9% of women advised to stop work because of swelling, fatigue, stress, or another reason, and in 13.4% of women advised to stop work because of labor, high blood pressure, or vaginal bleeding (P <.001). Among women advised to stop working in the first through seventh months of pregnancy, 91.7% (95% CI 88.8, 94.5) delivered at 36 or more weeks' gestation. CONCLUSION: Work cessation during pregnancy was commonly recommended in this population and was associated with clinical risk factors and adverse birth outcomes. For some women it resulted in a long period of work absence before delivery.

Prayer in medicine: a survey of primary care physicians.

Prayer and spirituality have been shown to have a significant impact on several health variables. Additionally, studies have shown that patients think prayer is important to their health. Very little research, however, has been done to determine primary care physicians' opinions regarding prayer and spirituality as it pertains to healthcare. We surveyed primary care physicians in Mississippi to assess their use of prayer in medical practice. Ninety-one percent of respondents considered prayer an important treatment modality, but 50.6% rarely or never discussed prayer with patients. Most who excluded prayer from clinical practice did so to avoid imposing their beliefs upon patients. A majority of primary care physicians in Mississippi recognize prayer as an important psychosocial variable in assessing and treating patients, but many are hesitant to incorporate this variable into the doctor-patient encounter.

Pregnancy intention: how PRAMS data can inform programs and policy. Pregnancy Risk Assessment Monitoring System.

BACKGROUND: In most states, the Pregnancy Risk Assessment Monitoring System (PRAMS) is the only source for state-specific, population-based data on the prevalence of unintended pregnancy among women having a live birth. These data can be used in a variety of ways to inform state policies and programs aimed at the prevention of unintended pregnancy. OBJECTIVES: This paper highlights the programmatic and policy development activities undertaken by three states in relation to unintended pregnancy as well as the role that PRAMS data played in those efforts. RESULTS: Georgia, Oklahoma, and Washington have used PRAMS data to gain support for program initiatives directed at unintended pregnancy, to promote policies aimed at either monitoring or reducing unintended pregnancy, and to acquire additional funds for related programs, such as family planning.

Effects of cationic charge on three-dimensional structures of intercalative complexes: structure of a bis-intercalated DNA complex solved by MAD phasing.

We characterize intercalative complexes as either "high charge" and "low charge". In low charge complexes, stacking interactions appear to dominate stability and structure. The dominance of stacking is evident in structures of daunomycin, nogalamycin, ethidium, and triostin A/echinomycin. By contrast in a DNA complex with the tetracationic metalloporphyrin CuTMPyP4 [copper (II) meso-tetra(N-methyl-4-pyridyl)porphyrin], electrostatic interactions appear to draw the porphyrin into the duplex interior, extending the DNA along its axis, and unstacking the DNA. Similarly, DNA complexes of tetracationic ditercalinium and tetracationic flexi-di show significant unstacking. Here we report x-ray structures of complexes of the tetracationic bis-intercalator D232 bound to DNA fragments d(CGTACG) and d(BrCGTABrCG). D232 is analogous to ditercalinium but with three methylene groups inserted between the piperidinium groups. The extension of the D232 linker allows it to sandwich four base pairs rather than two. In comparison to CuTMPyP4, flexi-di and ditercalinium, stacking interactions of D232 are significantly improved. We conclude that it is not sufficient to characterize intercalators simply by net charge. One anticipates strong electrostatic forces when cationic charge is focused to a small volume or region near DNA and so must consider the extent to which cationic charge is focused or distributed. In sum, ditercalinium, with a relatively short linker, focuses cationic charge more narrowly than does D232. So even though the net charges are equivalent, electrostatic charges are expected to be of greater structural significance in the ditercalinium complex than in the D232 complex.

Structures of yeast vesicle trafficking proteins.

In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.

Randomized, controlled trial of video self-instruction versus traditional CPR training.

STUDY OBJECTIVE: We conducted a prospective, randomized, controlled trial to test the hypothesis that a 34-minute video self-instruction (VSI) training program for adult CPR would yield comparable or better CPR performance than the current community standard, the American Heart Association Heartsaver course. METHODS: Incoming freshman medical students were randomly assigned to VSI or the Heartsaver CPR course. Two to 6 months after training, we tested subjects to determine their ability to perform CPR in a simulated cardiac arrest setting. Blinded observers used explicit criteria to assess our primary outcome, CPR performance skill. In addition, we assessed secondary outcomes including sequential performance of individual skills, ventilation and chest compression characteristics, and written tests of CPR-related knowledge and attitudes. RESULTS: VSI trainees displayed superior overall performance compared with traditional trainees. Twenty of 47 traditional trainees (43%) were judged not competent in their performance of CPR, compared with only 8 of 42 VSI trainees (19%; absolute difference, 24%; 95% confidence interval, 5% to 42%). CONCLUSION: In a group of incoming freshman medical students, we found that a half-hour of VSI resulted in superior overall CPR performance compared with that in traditional trainees. If validated by further research, VSI may provide a simple, quick, and inexpensive alternative to traditional CPR instruction for health care workers and, perhaps, the general population.

Oxygen administration during transport and recovery after outpatient surgery does not prevent episodic arterial desaturation.

STUDY OBJECTIVE: To compare the efficacy of two different oxygen (O2) delivery systems in preventing episodic arterial desaturation in the immediate postoperative period. STUDY DESIGN: Randomized, prospective, nonblinded comparison in patients. SETTING: Operating room and postanesthesia care unit (PACU) of a university outpatient surgery center. PATIENTS: 100 ASA status I and II adults, male and female, age greater than 18 years, undergoing outpatient surgical procedures not involving the upper airway. INTERVENTIONS: Group I received supplemental O2 administered by bag-valve-mask during transport, followed by 40% face shield in the PACU. Group 2 received supplemental O2 by nasal cannula at 4 L/min both during transport and during PACU stay. MEASUREMENTS AND MAIN RESULTS: Arterial O2 saturation (SpO2) was collected by computer from a recording pulse oximeter at 15-second intervals beginning before extubation and continuing until O2 administration was discontinued in PACU. Neither mode of therapy was successful in completely eliminating arterial desaturation defined as SpO2 less than 90%, nor was there any difference in efficacy between the two treatment groups. Group 1 had 8 patients desaturate on 9 different occasions (5 times during transport, 4 times in PACU). Group 2 had 5 patients desaturate on 9 different occasions (4 times during transport, 5 times in PACU). CONCLUSION: Routine O2 administration during transport and PACU stay did not abolish episodic desaturation, even in healthy patients undergoing minor surgical procedures. Given the marked difference in acquisition cost, it would appear that O2 administration by nasal cannula is a more cost-effective alternative for routine postoperative O2 administration in certain groups of patients undergoing general anesthesia for outpatient surgery.

Structure of DNA-porphyrin complex.

We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.

Clathrate hydrates are poor models of biomolecule hydration.

Clathrate hydrates form the basis of a general model of biomolecule hydration. In clathrate hydrate crystal structures, the size of hydrogen-bonded water rings is highly constrained to five members. The clathrate hydrate model predicts that the size of water rings near biomolecule surfaces is similarly constrained to five members. This report describes a test of this model of biomolecule hydration. We have demonstrated that five-membered water rings are not a general feature of protein or nucleic acid hydration. The clathrate hydrate model appears to be inappropriate for soluble biomolecules.

Asymmetry and dynamics in bis-intercalated DNA.

The bis-intercalator ditercalinium (NSC 366241), composed of two 7 H-pyridocarbazoles linked by a bis(ethylpiperidinium), binds to DNA with a binding constant greater than 10(7) M-1. One distinctive aspect of the 3-D X-ray structure of a DNA-ditercalinium complex is its asymmetry. We propose here that the activity of ditercalinium may be related to structural polymorphism and dynamic conversion between conformers. It was previously reported that activity is closely related to linker composition. Activity increases with increasing conformational restraints of the linker. We suggest these conformational restraints can lead to asymmetry in DNA complexes and that this asymmetry results directly in structural polymorphism. Using the Cambridge Structural Database (CSD) as a source of information about chemical fragments that are analogous to the linker of ditercalinium, we have explored the conformational space available to ditercalinium. The results indicate that the linker is highly constrained and that the DNA complex is intrinsically asymmetric. We propose a reasonable mechanism of ring reversal that is consistent with the conformations of analogous fragments within the CSD.

X-ray structure of a DNA decamer containing 7,8-dihydro-8-oxoguanine.

We have determined the x-ray structure of a DNA fragment containing 7,8-dihydro-8-oxoguanine (G(O)). The structure of the duplex form of d(CCAGOCGCTGG) has been determined to 1.6-A resolution. The results demonstrate that GO forms Watson-Crick base pairs with the opposite C and that G(O) is in the anti conformation. Structural perturbations induced by C.G(O)anti base pairs are subtle. The structure allows us to identify probable elements by which the DNA repair protein MutM recognizes its substrates. Hydrogen bond donors/acceptors within the major groove are the most likely element. In that groove, the pattern of hydrogen-bond donors/acceptors of C.G(O)anti is unique. Additional structural analysis indicates that conversion of G to G(O) would not significantly influence the glycosidic torsion preference of the nucleoside. There is no steric interaction of the 8-oxygen of G(O) with the phospho-deoxyribose backbone.

DNA distortion in bis-intercalated complexes.

The bis-intercalators Flexi-Di and ditercalinium are synthetic dimers that bis-intercalate into DNA and cause cell death in prokaryotes from futile and abortive repair of DNA. Each is composed of two 7H-pyridocarbazole units and a linker. Flexi-Di has a flexible spermine-like linker while ditercalinium has a rigid bis(ethylpiperidinium) linker. This report, describing the 2.5-A X-ray structure of Flexi-Di complexed with [d(BrCGCG)]2, appears to be the first report of a three-dimensional structure of a DNA complex with a bis-intercalator with a flexible linker. DNA complex formation with a ditercalinium analog having a flexible linker was not anticipated to yield unstacked and bent DNA as was observed in the previously reported ditercalinium.[d(CGCG)]2 complex. Surprisingly, the DNA in the Flexi-Di complex is bent to a degree exceeding that of the ditercalinium complex. A comparison of the DNA complexes of Flexi-Di and ditercalinium has allowed us to propose a mechanism by which these bis-intercalators distort DNA. We propose that this class of bis-intercalators pulls the internal base pairs into the major groove and pushes the external base pairs into the minor groove. The result is a bend toward the minor groove. It appears that hydrogen bonds between the linker and the internal guanines effectively pull the central base pairs of the complex out into the major groove. At the external regions of the complex, stacking interactions between the chromophores and terminal base pairs effectively push the terminal base pairs into the minor groove. The result of this push/pull combination is to bend the DNA.

Water ring structure at DNA interfaces: hydration and dynamics of DNA-anthracycline complexes.

In crystallographic structures of biological macromolecules, one can observe many hydration rings that originate at one water molecule, pass via hydrogen bonds through several others, and return to the original water molecule. Five-membered water rings have been thought to occur with greater frequency than other ring sizes. We describe a quantitative assessment of relationships between water ring size and frequency of occurrence in the vicinity of nucleic acid interfaces. This report focuses on low-temperature X-ray crystallographic structures of two anthracyclines, adriamycin (ADRI) and daunomycin (DAUN), bound to d(CGATCG) and on several DNA structures published previously by others. We have obtained excellent low-temperature (-160 degrees C, LT) X-ray intensity data for d(CGATCG)-adriamycin and d(CGATCG)-daunomycin with a multiwire area detector. The LTX-ray data sets contain 20% (daunomycin, LT-DAUN) and 35% (adriamycin, LT-ADRI) more reflections than were used to derive the original room-temperature (15 degrees C) structures [Frederick, C.A., Williams, L.D., Ughetto, G., van der Marel, G. A., van Boom, J.H., Rich, A., & Wang, A.H.-J. (1990) Biochemistry 29, 2538-2549]. The results show that five-membered water rings are not preferred over other ring sizes. This assessment is consistent with our observation of broad dispersion W-W-W angles (sigma = 20 degrees). In addition, we report that the thermal mobility, distinct from the static disorder, of the amino sugar of daunomycin and adriamycin is significantly greater than that of the rest of the complex. This mobility implies that if the central AT base pair is switched to a CG base pair, there should be a low energy cost in avoiding the guanine amino group. The energy difference (for the sugar-binding preference) between d(CGTACG) and d(CGCGCG) could be considerably less than 20 kcal/mol, a value proposed previously from computation.

Cleavage of tubulin by vanadate ion.

Vanadate is known to cleave proteins in a near-uv-dependent manner. We have found that vanadate will cleave alpha- and beta-tubulin upon photoirradiation (419 nm emission maxima) under conditions when tetravanadate, pentavanadate, and decavanadate are in solution. The reaction is independent of GTPMg or GDPMg, and cleavage occurs at two or more sites per chain. Cleavage was studied at pH 6.0 (2(N-morpholino)ethanesulfonic acid (Mes) and phosphate), pH 6.9 (piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes)), pH 7.0 (phosphate), and pH 8.0 (N-(2-hydroxyethyl)piperazine-N'-bis(2-ethanesulfonic acid) (Hepes) and phosphate). The concentration of vanadate oligomer species, as determined by 51V NMR, was correlated with the extent of cutting. In organic buffers, low pH and high vanadate concentration favored oligomer formation, especially tetra and decavanadate. In phosphate buffer at pH 7 and 8, decamer is more prevalent, and at pH 6, phosphate buffer appears to favor a different oligomer form, V', appearing at -582 ppm. Cleavage is best correlated with the presence of cyclic tetravanadate at pH 6.9 in Pipes buffer and the V' species at pH 6.0 in phosphate buffer. Cleavage efficiency is also affected by interactions of photoactivated vanadate species with organic buffer components. In phosphate buffer no photochemical degradation of vanadate species occurs. Analysis using sodium dodecyl sulfate (SDS) gel electrophoresis and western blotting showed that vanadate produced cleavage patterns and nonenzymatic cleavage patterns resulting from boiling tubulin in SDS sample buffer (J. J. Correia, L. D. Lipscomb, and S. Lobert, 1993, Arch. Biochem. Biophys. 300, 105-114) are not the same. Attempts to identify the locations of the vanadate cleavage sites on the protein through N-terminal sequencing was unsuccessful, apparently due to the presence of blocked amino groups. We conclude that tetravandate cleaves tubulin upon photoirradiation, that organic buffers can interact with vanadate oligomers upon photoirradiation, and that in phosphate buffer photocleavage is enhanced by an absence of photochemical degradation and a preference for forming photoactive vanadate oligomers. These results have general application to photoirradiation studies of any protein in the presence of vanadate.

Nondisulfide crosslinking and chemical cleavage of tubulin subunits: pH and temperature dependence.

Tubulin is known to be extremely unstable. The denaturation process partially involves irreversible aggregation, mediated by disulfide crosslinking. In addition, tubulin is known to undergo chemical cleavage during boiling in sodium dodecyl sulfate (SDS), a process that generates small peptides that have been mistaken for low molecular weight MAPs. Similar peptide cleavage has now been observed during two-dimensional denaturing isoelectric focusing-SDS-polyacrylamide gel electrophoresis. This phenomenon has complicated interpretation of limited proteolysis studies of tubulin by subtilisin. In an effort to avoid this problem we have undertaken a detailed study of the solution conditions that promote chemical cleavage of tubulin. The cleavage reaction is found to be strongly pH, time, and temperature dependent. Nondenatured and denatured tubulin is susceptible to peptide cleavage, suggesting that primary structure is more important than secondary structure in selection of susceptible bonds. After transfer of cleavage products from an SDS gel to a polyvinylidene difluoride membrane, amino acid sequencing has confirmed cleavage at Asp-Pro bonds, at position 306 in alpha-tubulin and at position 304 in beta-tubulin. We also infer cleavage at the only additional Asp-Pro peptide bond located at position 31 in beta-tubulin. Heat-induced cleavage at Asp-Pro accounts for 5 of the 13 bands observed on SDS gels. In addition, a minor alpha-tubulin band has been sequenced from a two-dimensional gel, corresponds to cleavage at Asp-Cys located at alpha-tubulin position 200, and accounts for two additional bands observed on SDS gels. Under nondenaturing and nonpolymerizing conditions tubulin undergoes extensive intermolecular, disulfide crosslinking. At elevated temperatures and high pH, a small fraction of the crosslinking is not reduced by beta-mercaptoethanol. Disulfide-crosslinked aggregates are not suspected because carboxymethylation of tubulin does not prevent their formation. Lysinoalanine has been found by amino acid analysis and thus covalent lysine-dehydroalanine crosslinks are suspected. Dehydroalanine is formed by beta-elimination at serine and thus the presence of lysinoalanine is consistent with cleavage at Gly-Ser peptides, the most unstable serine peptide bond, and accounts for most of the remaining cleavage data.