Objective Improvements in clinical pain care have not matched advances in scientific knowledge, and innovations in medical education are needed. Several streams of evidence indicate that pain education needs to address both the affective and cognitive dimensions of pain. Our aim was to design and deliver a new course in pain establishing foundation-level knowledge while comprehensively addressing the emotional development needs in this area. Setting 118 first year medical students at Johns Hopkins School of Medicine. Outcome measures Performance was measured by multiple choice tests of pain knowledge, attendance, reflective pain portfolios and satisfaction measures. Results Domains of competence in pain knowledge included central and peripheral pain signaling, pharmacological management of pain with standard analgesic medications, neuromodulating agents and opioids; cancer pain, musculoskeletal pain, nociceptive, inflammatory, neuropathic, geriatric, and pediatric pain. Socio-emotional development (portfolio) work focused on increasing awareness of pain affect in self and others and enhancing the commitment to excellence in pain care. Reflections included observations on a brief pain experience (cold pressor test), the multi-dimensionality of pain, the role of empathy and compassion in medical care, the positive characteristics of pain-care role models, the complex feelings engendered by pain and addiction including frustration and disappointment, and aspirations and commitments in clinical medicine. The students completing feedback expressed high levels of interest in pain medicine as a result of the course. Discussion We conclude that a four-day pain course incorporating sessions with pain- specialists, pain medicine knowledge, and design-built elements to strengthen emotional skills is an effective educational approach.
Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the acetyl-CoA carboxylase. Biotinyl-5'-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5'-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5'-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5'-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.
SlOOB is a Ca2+-binding protein known to be a noncovalently associated dimer, S IOOB(PP), at high concentrations (0.2-3.0 mM) under reducing conditions. The solution structure of apo-SlOOB(PP) shows that the subunits associate in an antiparallel manner to form a tightly packed hydrophobic core at the dimer interface involving six of eight helices and the C-terminal loop (Drohat AC, Amburgey JC, Abildgaard F, Starich MR, Baldisseri D, Weber DJ. 1996. Solution structure of rat apo-S lOOB(PP) as determined by NMR spectroscopy. Biochemistry 351 1577-1 1588).The C-terminal loop, however, is also known to participate in the binding of SlOOB to target proteins, so its participation in the dimer interface raises questions as to the physiological relevance of dimeric SlOOB(PP). Therefore, we investigated the oligomerization state of SlOOB at low concentrations (1-10,OOO nM) using large-zone analytical gel filtration chromatography with %-labeled S100B. We found that SlOOB exists (>99%) as a non-covalently associated dimer, SlOOB(PP), at 1 nM subunit concentration (500 pM dimer) in the presence or absence of saturating levels of Ca2+, which implies a dissociation constant in the picomolar range or lower. These results demonstrate for the first time that in reducing environments and at physiological concentrations, SlOOB exists as dimeric SlOOB(PP) in the presence or absence of Ca2+, and that the non-covalent dimer is most likely the form of SlOOB presented to target proteins.
The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme. BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase. BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes. Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA. Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change. The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes. Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed. Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct.
New drugs and molecular targets are needed against Trypanosoma brucei, the protozoan that causes African sleeping sickness. Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione), a traditional antifungal agent, and 11 analogs were tested against T. brucei in vitro. The greatest activity was conferred by electron-withdrawing groups in the 8 position of the tryptanthrin ring system; the most potent compound had a 50% effective concentration of 0.40 M.Members of the Trypanosoma brucei species are flagellated protozoa that are transmitted by tsetse flies. They cause sleeping sickness in humans and a related disease in cattle. The characteristic meningoencephalitis of African sleeping sickness is fatal if it is not treated, and currently available drugs are limited by toxicity and growing parasite resistance (7,9,12). Unfortunately, in recent years there has been a dramatic and devastating resurgence of sleeping sickness (14). The need for safe and effective new antitrypanosomal agents is pressing.Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione; in Fig. 1, X is H in tryptanthrin) is a weakly basic alkaloid found in a number of plant species (11). This alkaloid is unusual in that its synthesis was described a half century before it was discovered as a natural product (6). Tryptanthrin is the active principal of a traditional Japanese herbal remedy for fungal infections (8). Subsequent studies extended the spectrum of antimicrobial activity to include bacteria (10, 11), particularly Mycobacterium tuberculosis (1). The activity of tryptanthrin against this intracellular organism prompted us to evaluate tryptanthrin and a series of derivatives of tryptanthrin against intracellular parasites, including Plasmodium falciparum, which causes malaria (13). In this report we describe the activities of a series of substituted tryptanthrins and 4-azatryptanthrins (Fig. 1) against Trypanosoma brucei, an extracellular protozoan parasite.Tryptanthrins. Tryptanthrin compounds were synthesized as described by Baker and Mitscher (1) and were characterized by their infrared and mass spectra. Stock solutions were prepared in dimethyl sulfoxide (99.8%; 27,685-5; Aldrich) and were then serially diluted into culture medium.Assay. Bloodstream-form T. brucei (MiTat 1.2, strain 427) organisms were grown axenically (5) at 37°C in HEPES-buffered Iscove's modified Dulbecco's medium that did not contain phenol red but that was supplemented with glutamate, hypoxanthine, cysteine, thymidine, sodium pyruvate, mercaptoethanol, bathocuproinedisulfonate, 10% Serum Plus, and 10% heat-inactivated fetal bovine serum, as described previously (2). Ten concentrations of each tryptanthrin were assayed in quadruplicate. Exponentially growing cells were incubated in 96-well plates (Falcon no. 3072 plates; Becton Dickinson) with or without test compound for 20 h and were then lysed and incubated for 3 to 6 h with p-nitrophenol phosphate. The acid phosphatase activity was determined, and 50% effective concentrations (EC 50 s) were obtained from curves fit to the da...
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