Reply: a sanguine approach to cannabis
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Journal Article↗
Biomedical subjects
D Baker
Publications and source records attributed to D Baker.
Unrelated donors selected prospectively by block-matching have superior bone marrow transplant outcome.
Previous retrospective studies have demonstrated improved outcome in patients whose donors were matched for non-HLA markers in the MHC as well as for HLA genes. Forty patients receiving transplants from unrelated donors were typed prospectively for HLA and non-HLA markers. Non-HLA markers near HLA-B (beta-block markers) and in the DRB1 introns (delta-block markers) were used to assess MHC match between donors and recipient. Patients whose donors were matched at the beta- and delta-blocks had improved event free survival (63%) compared to patients whose donors were mismatched at one or both blocks (25%) (p < 0.05). Patients whose donors were matched at the beta-block had significantly less severe acute graft versus host disease (p < 0.05). In order to investigate the basis for improved outcome block matching was correlated with HLA matching as determined by DNA sequencing. Beta-block matching was highly correlated with matching for exons 2 and 3 of HLA-B but less so for HLA-C. Delta-block matching was highly correlated with matching for exon 2 of HLA DRB1. It is concluded that matching for non-HLA markers in the MHC improves matching for HLA genes. Further studies are required to determine whether matching for non-HLA markers improves outcome to a greater extent than matching for the HLA genes alone.
Adolescent↗
De novo protein structure determination using sparse NMR data.
We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with approximately 1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined.
Humans↗
Critical role of beta-hairpin formation in protein G folding.
Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the approximately 60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed beta-hairpins. We demonstrate that, as in the case of protein L, one of the two beta-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second beta-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain-main chain hydrogen bond in the second beta-turn slows the folding rate approximately 20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact beta-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated beta-hairpins.
Amino Acid Substitution↗
High-dose carmustine, thiotepa and etoposide followed by autologous bone marrow rescue for the treatment of high risk central nervous system tumors.
Forty-two patients (29 newly diagnosed) with high grade gliomas (n = 37), medulloblastoma (n = 2) or non-biopsied tumors (n = 3) with supratentorial (n = 24), brain stem (n = 11), posterior fossa (n = 5) or spinal (n = 2) location were eligible for this study with adequate organ function and no bone marrow tumor infiltration. Median patient age was 12.2 years (range, 0.7-46.8). A total of 600 mg/m2 BCNU, 900 mg/m2 thiotepa and 1500 or 750 mg/m2 etoposide (VP-16) was administered followed by autologous bone marrow reinfusion (ABMR). Twenty-one newly diagnosed patients received local irradiation (RT) post ABMR. Nine early deaths were observed (21%), as well as one secondary graft failure. Half of the patients aged 18 years or older experienced toxic deaths, whereas only 15% of patients younger than 18 years experienced toxic death (P = 0.05). Of 25 evaluable newly diagnosed patients, 20% achieved complete remission (CR) and 4% partial remission (PR), while 28% remained in continuing complete remission (CCR) and 44% remained with stable disease prior to RT. Of eight evaluable patients with recurrent disease, one achieved CR and two PR, while one remained in CCR and four with stable disease for 1 to 110.2 months. Overall survival was 36%, 24% and 17% at 1, 2 and 3 years following ABMR, with three newly diagnosed patients and one patient treated for recurrent disease being alive, without disease progression 64.4, 67.0, 86.3 and 110.2 months after ABMR, respectively. The combination of high-dose BCNU/ thiotepa/VP-16 has substantial toxicity but definite activity for high risk CNS tumors. Similar protocols with lower toxicity merit further evaluation in both newly diagnosed and recurrent CNS tumors.
Adolescent↗
Immuno- and genetic therapy in autoimmune diseases.
Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.
Animals↗
Rhytidectomy with lateral SMASectomy.
Since 1992 an alternative to formal elevation of the superficial fascia is to perform a lateral SMASectomy, removing a portion of the SMAS in the region directly overlying the anterior edge of the parotid gland. Excision of the superficial fascia in this region secures mobile anterior SMAS to the fixed portion of the superficial fascia overlying the parotid. The direction in which the SMASectomy is performed is oriented so that the vectors of elevation following SMAS closure lie perpendicular to the nasolabial fold, thereby producing improvement not only of the nasolabial fold but also of the jowl and jawline. This method represents a rapid, safe, and reproducible operation, allowing the surgeon the versatility obtained with formal SMAS flap undermining while producing both the safety and rapidity of SMAS plication.
Cheek↗
Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG-binding domain of protein L from Peptostreptococcus magnus.
The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1. 7, 2.3 and 1.8 A, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites.
Alanine↗
Pharmacological characterization of phospholipid growth-factor receptors.
The phospholipid growth-factor (PLGE) terminology is proposed to describe a group of endogenous glycerol- and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus-coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl-glycerophosphate, cyclic-phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA-activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT-PCR results reinforce the coexpression of PSP24 alpha and different members of the EDG1-7 receptors in the same cell. Stable heterologous expression of the PSP24 alpha, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose-response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.
Animals↗
Human immunodeficiency virus type 1 mutations selected in patients failing efavirenz combination therapy.
Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenz-zidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.
Alkynes↗
Domiciliary visits to the old and the mentally ill: how valuable?
In the early days of the British National Health Service, domiciliary visits were a continuation of the tradition whereby general practitioners (GPs) met consultants in the patient's home. The nature of domiciliary visits, which still attract a special fee, has since changed. We analysed the effectiveness of all domiciliary visits undertaken in a NHS trust providing primary care, mental health and elderly care services to a population of 470,000. Data were obtained from domiciliary visit claim forms and from questionnaires completed by the consultant, the referring GP and consultant peer reviewers. The largest number of visits (total 234) was in geriatric medicine 48.9%, followed by old-age psychiatry 44.9%. Geriatric medicine was more likely than psychiatry to admit patients to hospital (19%) after a visit. All domiciliary visits in old-age psychiatry were done during the day (9 am to 5 pm). Only 2% of GPs stated that they attended any of the domiciliary visits; almost all thought that the outcome of domiciliary visits was of value. Old-age-psychiatry peer reviewers believed that all visits in that specialty were appropriate; in geriatric medicine this figure was 77% and in other psychiatric specialties 65%. The findings indicate that domiciliary visits were not being used routinely as a pathway to hospital admission, though they were often used to expedite admission or gain a quick consultant opinion; the visits were valued by GPs. The practice of domiciliary visits differs greatly from the definition in NHS terms and conditions of service. One or other should be altered.
Aged↗
HIV/AIDS consultations.
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Acquired Immunodeficiency Syndrome↗
The use of time step simulations and difference equations (TSSADEQ) in modeling heparin pharmacokinetics.
A model in which the clearance of heparin requires the binding of heparin to a finite and regenerated pool of binding was constructed using time step simulations and difference equations (TSSADEQ). A simulation of a heparin i.v. bolus demonstrated a dose-dependent, triphasic pharmacokinetic curve with (1) an initial log-linear phase representing first-order association of heparin with binding sites, (2) an intermediate plateau phase representing constant regeneration of heparin binding sites, and (3) a terminal log-linear phase occurring when the quantity of regenerated sites exceeded the remaining heparin. Sensitivity analysis based on the literature produced estimates of the k at 1.39 to 2.77 h-1, the pool of binding sites of 50 units/kg, and the regeneration rate of 15 to 20 unit/kg/h--virtually identical to the empirically derived guidelines for the bolus size and infusion rate for unfractionated heparin. A pilot study of bolus dosing of unfractionated heparin in normal volunteers confirmed the model.
Adult↗
Structural transitions in the protein L denatured state ensemble.
We use a broad array of biophysical methods to probe the extent of structure and time scale of structural transitions in the protein L denatured state ensemble. Measurement of amide proton exchange protection during the first several milliseconds following initiation of refolding in 0.4 M sodium sulfate revealed weak protection in the first beta-hairpin and helix. A tryptophan residue was introduced into the first beta-hairpin to probe the extent of structure formation in this part of the protein; the intrinsic fluorescence of this tryptophan was found to deviate from that expected given its local sequence context in 2-3 M guanidine, suggesting some partial ordering of this region in the unfolded state ensemble. To further probe this partial ordering, dansyl groups were introduced via cysteine residues at three sites in the protein. It was found that fluorescence energy transfer from the introduced tryptophan to the dansyl groups decreased dramatically upon unfolding. Stopped-flow fluorescence studies showed that the recovery of dansyl fluorescence upon refolding occurred on a submillisecond time scale. To probe the interactions responsible for the residual structure observed in the denatured state ensemble, the conformation of a peptide corresponding to the first beta-hairpin and helix of protein L was studied using circular dichroism spectroscopy and compared to that of full-length protein L and previously characterized peptides corresponding to the isolated helix and second beta-hairpin.
Amino Acid Sequence↗
Prediction of protein-folding mechanisms from free-energy landscapes derived from native structures.
Guided by recent experimental results suggesting that protein-folding rates and mechanisms are determined largely by native-state topology, we develop a simple model for protein folding free-energy landscapes based on native-state structures. The configurations considered by the model contain one or two contiguous stretches of residues ordered as in the native structure with all other residues completely disordered; the free energy of each configuration is the difference between the entropic cost of ordering the residues, which depends on the total number of residues ordered and the length of the loop between the two ordered segments, and the favorable attractive interactions, which are taken to be proportional to the total surface area buried by the ordered residues in the native structure. Folding kinetics are modeled by allowing only one residue to become ordered/disordered at a time, and a rigorous and exact method is used to identify free-energy maxima on the lowest free-energy paths connecting the fully disordered and fully ordered configurations. The distribution of structure in these free-energy maxima, which comprise the transition-state ensemble in the model, are reasonably consistent with experimental data on the folding transition state for five of seven proteins studied. Thus, the model appears to capture, at least in part, the basic physics underlying protein folding and the aspects of native-state topology that determine protein-folding mechanisms.
Models, Theoretical↗
Hierarchy of structure loss in MD simulations of src SH3 domain unfolding.
To complement experimental studies of the src SH3 domain folding, we studied 30 independent, high-temperature, molecular dynamics simulations of src SH3 domain unfolding. These trajectories were observed to differ widely from each other. Thus, rather than analyzing individual trajectories, we sought to identify the recurrent features of the high-temperature unfolding process. The conformations from all simulations were combined and then divided into groups based on the number of native contacts. Average occupancies of each side-chain hydrophobic contact and hydrogen bond in the protein were then determined. In the symmetric funnel limit, the occupancies of all contacts should decrease in concert with the loss in total number of native contacts. If there is a lack of symmetry or hierarchy to the unfolding process, the occupancies of some contacts should decrease more slowly, and others more rapidly. Despite the heterogeneity of the individual trajectories, the ensemble averaging revealed an order to the unfolding process: contacts between the N and C-terminal strands are the first to disappear, whereas contacts within the distal beta-hairpin and a hydrogen-bonding network involving the distal loop beta-turn and the diverging turn persist well after the majority of the native contacts are lost. This hierarchy of events resembles but is somewhat less pronounced than that observed in our experimental studies of the folding of src SH3 domain.
Computer Simulation↗
Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins.
We describe the development of a scoring function based on the decomposition P(structure/sequence) proportional to P(sequence/structure) *P(structure), which outperforms previous scoring functions in correctly identifying native-like protein structures in large ensembles of compact decoys. The first term captures sequence-dependent features of protein structures, such as the burial of hydrophobic residues in the core, the second term, universal sequence-independent features, such as the assembly of beta-strands into beta-sheets. The efficacies of a wide variety of sequence-dependent and sequence-independent features of protein structures for recognizing native-like structures were systematically evaluated using ensembles of approximately 30,000 compact conformations with fixed secondary structure for each of 17 small protein domains. The best results were obtained using a core scoring function with P(sequence/structure) parameterized similarly to our previous work (Simons et al., J Mol Biol 1997;268:209-225] and P(structure) focused on secondary structure packing preferences; while several additional features had some discriminatory power on their own, they did not provide any additional discriminatory power when combined with the core scoring function. Our results, on both the training set and the independent decoy set of Park and Levitt (J Mol Biol 1996;258:367-392), suggest that this scoring function should contribute to the prediction of tertiary structure from knowledge of sequence and secondary structure.
Models, Statistical↗
Ab initio protein structure prediction of CASP III targets using ROSETTA.
To generate structures consistent with both the local and nonlocal interactions responsible for protein stability, 3 and 9 residue fragments of known structures with local sequences similar to the target sequence were assembled into complete tertiary structures using a Monte Carlo simulated annealing procedure (Simons et al., J Mol Biol 1997; 268:209-225). The scoring function used in the simulated annealing procedure consists of sequence-dependent terms representing hydrophobic burial and specific pair interactions such as electrostatics and disulfide bonding and sequence-independent terms representing hard sphere packing, alpha-helix and beta-strand packing, and the collection of beta-strands in beta-sheets (Simons et al., Proteins 1999;34:82-95). For each of 21 small, ab initio targets, 1,200 final structures were constructed, each the result of 100,000 attempted fragment substitutions. The five structures submitted for the CASP III experiment were chosen from the approximately 25 structures with the lowest scores in the broadest minima (assessed through the number of structural neighbors; Shortle et al., Proc Natl Acad Sci USA 1998;95:1158-1162). The results were encouraging: highlights of the predictions include a 99-residue segment for MarA with an rmsd of 6.4 A to the native structure, a 95-residue (full length) prediction for the EH2 domain of EPS15 with an rmsd of 6.0 A, a 75-residue segment of DNAB helicase with an rmsd of 4.7 A, and a 67-residue segment of ribosomal protein L30 with an rmsd of 3.8 A. These results suggest that ab initio methods may soon become useful for low-resolution structure prediction for proteins that lack a close homologue of known structure.
Algorithms↗