In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin
degradation results in loss of axonal function and eventual axonal degeneration.
Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to
remyelination of denuded axons occurs regularly in early stages of MS but halts as
the pathology transitions into progressive MS. Pharmacological potentiation of
endogenous OPC maturation and remyelination is now recognized as a promising
therapeutic approach for MS. In this study, we analyzed the effects of modulating the
Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective
inhibitors of ROCK, on the transformation of OPCs into mature, myelinating
oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent
and human origin, that ROCK inhibition in OPCs results in a significant generation of
branches and cell processes in early differentiation stages, followed by accelerated
production of myelin protein as an indication of advanced maturation. Furthermore,
inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons
and remyelination in rat cerebellar tissue explants previously demyelinated with
lysolecithin. Our findings indicate that by direct inhibition of this signaling
molecule, the OPC differentiation program is activated resulting in morphological and
functional cell maturation, myelin formation, and regeneration. Altogether, we show
evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the
induction of remyelination in demyelinating pathologies.
cytoskeletal dynamics; human OPCs; maturation; myelin regeneration; oligodendrocytes; ROCK inhibition
The crystal structure of E. coli aspartate α-decarboxylase Asn72Ala reveals the structure of the previously unresolved C-terminus.
The crystal structure of the Asn72Ala site-directed mutant of Escherichia coli aspartate α-decarboxylase (ADC) has been determined at 1.7 Å resolution. The refined structure is consistent with the presence of a hydrolysis product serine in the active site in place of the pyruvoyl group required for catalysis, which suggests that the role of Asn72 is to protect the ester formed during ADC activation from hydrolysis. In previously determined structures of activated ADC, including the wild type and other site-directed mutants, the C-terminal region of the protein is disordered, but in the Asn72Ala mutant these residues are ordered owing to an interaction with the active site of the neighbouring symmetry-related multimer.
pantothenate; pyruvoyl-dependent; autocatalytic activation; crystallographic artefacts
Threonine 57 is identified as the key residue required for the post-translational activation of E. coli aspartate decarboxylase. The crystal structure of the site-directed mutant T57V is reported.
Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.
aspartate decarboxylase; post-translational modification; amino-acid-derived cofactors; pyruvoyl-dependent
Plasmacytomas are malignant proliferations of plasma cells which can occur with different plasma cell dyscrasias. Solitary plasmacytomas of bone or extraosseous plasmacytomas, depending on the tissue of origin, develop in isolation without systemic manifestations of multiple myeloma. Three cases of cranial plasmacytomas are described, two in patients with multiple myeloma and one extraosseous plasmacytoma. Management options are discussed.
Cranial; Plasmacytoma; Multiple myeloma; Treatment
A series of aryl piperazinyl ureas that act as covalent
of fatty acid amide hydrolase (FAAH) is described. A potent and selective
(does not inhibit FAAH-2) member of this class, JNJ-40355003, was
found to elevate the plasma levels of three fatty acid amides: anandamide,
oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog,
and cynomolgous monkey. The elevation of the levels of these lipids
in the plasma of monkeys suggests that FAAH-2 may not play a significant
role in regulating plasma levels of fatty acid ethanolamides in primates.
FAAH; urea; ethanolamides; FAAH-2; enzyme; anandamide
The outcome in patients with chronic myeloid leukemia (CML) has dramatically improved over the last decade due to the widespread use of novel tyrosine kinase inhibitors such as imatinib. As overall survival has improved, the number of women with CML that wish to become pregnant has increased. As such, attending physicians are faced with a dilemma - continue life-prolonging medication to treat the cancer, or interrupt its use due to its potential teratogenicity. Herein we describe 2 CML patients that gave birth. Case 1 was managed via substitution of imatinib with interferon. The patient’s child underwent genetic evaluation at age 3 years, achieved normal developmental milestones, and despite being shorter than his peers was proportional. In terms of morphology, the child had clinodactyly, short fifth fingers, and slightly downward slanting palpebral fissures, but otherwise appeared normal. In case 2 imatinib was continued throughout the pregnancy. This patient’s child underwent postpartum evaluation by a geneticist and was observed to be morphologically normal, except for clinodactyly and low-set ears.
Conflict of interest:None declared.
Imatinib; Tyrosine kinase inhibitors; Chronic myeloid leukemia; Pregnancy
► PanZ is required for formation of the pyruvoyl-cofactor in PanD (ADC) in vivo. ► PanZ and PanD interact with nanomolar affinity. ► Interaction of PanZ and PanD is dependent upon coenzyme A. ► PanZ and PanD form a heterooctameric complex which binds four molecules of CoA.
The existence of a fifth essential protein for pantothenate biosynthesis in some enteric bacteria has recently been reported by Stuecker et al.  and Nozaki et al. (in press) . This protein, PanZ, catalyses the activation of the PanD zymogen to form ADC and is essential for prototrophic growth. In this paper, we characterise the interaction of PanZ with coenzyme A and a constitutively inactive mutant of PanD using a combination of isothermal titration calorimetry and mass spectrometry. These approaches reveal that the two proteins interact with nanomolar affinity in a CoA-dependent fashion to form a heterooctameric complex.
Pantothenate; Vitamin biosynthesis; Pyruvoyl-cofactor; Post-translational modification
In bacteria, β-alanine is formed via the action of l-aspartate α-decarboxylase (PanD) which is one of the small class of pyruvoyl-dependent enzymes. The pyruvoyl cofactor in these enzymes is formed via the intramolecular rearrangement of a serine residue in the peptide backbone leading to chain cleavage and formation of the covalently-bound cofactor from the serine residue. This reaction was previously thought to be uncatalysed. Here we show that in Escherichia coli, PanD is activated by the putative acetyltransferase YhhK, subsequently termed PanZ. Activation of PanD both in vivo and in vitro is PanZ-dependent. PanZ binds to PanD, and we demonstrate that a PanZ(N45A) site-directed mutant is unable to enhance cleavage of the proenzyme PanD despite retaining affinity for PanD. This suggests that the putative acetyltransferases domain of PanZ may be responsible for activation to enhance the processing of PanD. Although panD is conserved among most bacteria, the panZ gene is conserved only in E. coli-related enterobacterial species including Shigella, Salmonella, Klebsiella and Yersinia. These bacteria are found predominantly in the gut flora where pantothenate is abundant and regulation of PanD by PanZ allows these organisms to closely regulate production of β-alanine and hence pantothenate in response to metabolic demand.
ADC; β-alanine; coenzyme A; PanD; pantothenate; YhhK
Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resembles that of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed EM reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms, a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of simple nicked circular product rather than complex networks of partially exchanged substrates.
DNA repair; recombination; RecA; electron microscopy; X-ray crystallography
We describe the case of a patient with small cell lung cancer and dual paraneoplastic syndromes involving adrenocorticotropic hormone and calcitonin. To the best of our knowledge, dual paraneoplastic syndromes involving these two hormones have not been previously reported in the literature.
A 74-year-old Caucasian woman presented with a left hilar mass and metastatic disease in the liver and right adrenal gland. The patient complained only of intermittent diarrhea. Her laboratory values exhibited metabolic alkalosis with hypokalemia, hypocalcemia, hypomagnesemia, hypophosphatemia, and hyperglycemia.
We discuss the work-up and treatment of the patient's unusual laboratory presentation with two concurrent paraneoplastic syndromes.
This case report describes the occurrence of bilateral myositis ossificans in the rectus femoris muscles of a young Gaelic football player with a long history of recurrent bilateral thigh strain. In each case, clinical diagnosis was followed up with biochemical profiling and sonographic investigations. Management consisted of rest from elite level competition and intense rehabilitation to address any potential risk factors for rectus femoris strain. A 4-week course of acetic acid iontophoresis was administered to the first myositis ossificans lesion on the left thigh; however, as this did not result in any significant changes to the lesion’s dimensions, it was not used on the contralateral lesion. The athlete returned to full sporting capacity 4 months after the first lesion was diagnosed. A 13-month follow-up showed that the athlete continued to play to full capacity with no recurrence of injury.
Anthocyanins and their aglycone anthocyanidins are pigmented flavonoids found in significant amounts in many commonly consumed foods. They exhibit a complex chemistry in aqueous solution, which makes it difficult to study their chemistry under physiological conditions. Here we used a gel electrophoresis assay employing supercoiled DNA plasmid to examine the ability of these compounds (1) to intercalate DNA, (2) to inhibit human topoisomerase I through both inhibition of plasmid relaxation activity (catalytic inhibition) and stabilization of the cleavable DNA-topoisomerase complex (poisoning), and (3) to inhibit or enhance oxidative single-strand DNA nicking. We found no evidence of DNA intercalation by anthocyan(id)ins in the physiological pH range for any of the compounds used in this study—cyanidin chloride, cyanidin 3-O-glucoside, cyanidin 3,5-O-diglucoside, malvidin 3-O-glucoside and luteolinidin chloride. The anthocyanins inhibited topoisomerase relaxation activity only at high concentrations (> 50 μM) and we could find no evidence of topoisomerase I cleavable complex stabilization by these compounds. However, we observed that all of the anthocyan(id)ins used in this study were capable of inducing significant oxidative DNA strand cleavage (nicking) in the presence of 1 mM DTT (dithiothreitol), while the free radical scavenger, DMSO, at concentrations typically used in similar studies, completely inhibited DNA nicking. Finally, we propose a mechanism to explain the anthocyan(id)in induced oxidative DNA cleavage observed under our experimental conditions.
Anthocyanin; topoisomerase I; oxidation; DNA; intercalation; strand-cleavage; DNA nicking
Developing general strategies for inhibition of protein–protein interactions is a key challenge in chemical biology. Herein we describe oligobenzamide inhibitors of the p53–hDM2 protein–protein interaction.
Oligobenzamide inhibitors of the p53–hDM2 protein–protein interaction are described.
We describe a case in which reflection of a palatal flap for removal of a mesiodens is presented as the triggering factor for bradycardia caused by stimulation of the trigeminocardiac reflex. The management of the case, as well as the reflex arc, is discussed.
Trigeminocardiac reflex; Bradycardia
The purpose of this study was to compare the effect of oral versus submucosal meperidine on the behavior of pediatric dental patients. Twenty charts (10 in each group) were retrospectively reviewed. The groups were matched for age and weight. Presedation and postsedation behavior was rated. No difference was found in the increase in cooperation between the oral and the submucosal meperidine groups. While no difference was found between the 2 groups, a larger prospective study is needed to confirm these findings.
Objective: To assess the attitudes of undergraduate chiropractic and osteopathic students at Royal Melbourne Institute of Technology (RMIT) in 1992 on the education they are receiving and on the effectiveness of chiropractic and osteopathic care.
Design: Cross-sectional descriptive survey.
Participants: Undergraduate chiropractic and osteopathic students enrolled at RMIT School of Chiropractic and Osteopathy in 1992.
Results: This study surveyed 272 students, 196 who were chiropractic students and 76 who were osteopathic students from RMIT School of Chiropractic and Osteopathy in Melbourne, Australia. The students that responded represented 73.4% of chiropractic students and 85.4% of osteopathic students currently enrolled in their respective courses. Chiropractic and osteopathic students entered their respective courses from non-chiropractic/non-osteopathic families. More chiropractic students than osteopathic students (1.3:1.0) had their respective course as their first choice when applying for tertiary education. A majority (95.8 chiropractic students and 94.8% osteopathic students) of both groups surveyed were pleased with their choice of course. Students from both disciplines held considerable respect for each other in the care of certain conditions, but did not see the other profession’s care as effective as their own. A greater percentage of osteopathic students believed there was sufficient difference between chiropractic and osteopathy to justify two separate professions (57.6% compared to 97.2%).
Discussion: High quality education is a major aim in our schools and colleges. For this standard to be maintained it requires continual re-evaluation and assessment. Surveys such as this should be performed regularly as a method of evaluating student attitude and how these attitudes change during the course. This would also allow administrators to determine whether they are achieving their academic intentions. An immediate follow up survey asking the same questions is suggested to ascertain whether the same attitudes exist today.
Chiropractic; osteopathic medicine; education; students; attitude
We have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding SH2 domain from the eukaryotic cell signalling protein Grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. These investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo.
cell signaling; phosphohistidine; phosphotyrosine; protein modifications; synthetic analogues