Colchicine: an ancient drug with novel applications.

Colchicine is a treatment for gout that has been used for more than a millennium. It is the treatment of choice for familial Mediterranean fever and its associated complication, amyloidosis. The 2009 U.S. Food and Drug Administration approval of colchicine as a new drug had research consequences. Recent investigations with large cohorts of patients with gout who have been taking colchicine for years have demonstrated novel applications within oncology, immunology, cardiology and dermatology. Some emerging dermatological uses include the treatment of epidermolysis bullosa acquisita, leucocytoclastic vasculitis, aphthous stomatitis and others. In this work we relate the history and the new horizon of this ancient medicine.

p53 is associated with cellular microtubules and is transported to the nucleus by dynein.

Here we show that p53 protein is physically associated with tubulin in vivo and in vitro, and that it localizes to cellular microtubules. Treatment with vincristine or paclitaxel before DNA-damage or before leptomycin B treatment reduces nuclear accumulation of p53 and expression of mdm2 and p21. Overexpression of dynamitin or microinjection of anti-dynein antibody before DNA damage abrogates nuclear accumulation of p53. Our results indicate that transport of p53 along microtubules is dynein-dependent. The first 25 amino acids of p53 contain the residues that are essential for binding to microtubules. We propose that functional microtubules and the dynein motor protein participate in transport of p53 and facilitate its accumulation in the nucleus after DNA damage.

HIV-1 rev depolymerizes microtubules to form stable bilayered rings.

We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44-49 nm in external diameter, 3.4-4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg(2+) and is inhibited by maytansine. The interaction involves the NH(2)-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH(2)-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection.

Cyclic AMP-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by antimitotic agents.

The stimulation of steroidogenesis by antimitotic drugs has been studied in wild-type (Y-1) and cAMP-dependent protein kinase-deficient (kin-8) mouse adrenal tumor cell lines. Unlike some other cells, Y-1 cells do not increase their cAMP output upon exposure to antimitotic drugs such as colchicine, vinblastine or podophyllotoxin, which readily increase steroidogenesis. Moreover, no increase in cAMP can be detected over an extended time span. Stabilization of tubulin polymers by taxol or high concentrations of vinblastine blocks ACTH-, cholera toxin- or colchicine-stimulated steroidogenesis without major effects on cAMP levels. Colchicine and podophyllotoxin stimulate steroidogenesis in the cAMP-dependent protein kinase-deficient mutant to the same degree as in the wild-type Y-1 cells, although absolute steroid yields are lower in the mutant cells. We suggest that the antimitotic agents stimulate adrenal steroidogenesis by a cAMP-independent pathway that may involve facilitation of cholesterol access to the mitochondrion.

Podophyllotoxin, steganacin and combretastatin: natural products that bind at the colchicine site of tubulin.

A large number of antimicrotubule agents are known that bind to tubulin in vitro and disrupt microtubule assembly in vitro and in vivo. Many of these agents bind to the same site on the tubulin molecule, as does colchicine. Of these, the natural products podophyllotoxin, steganacin and combretastatin are the subjects of this review. For each of these, the chemistry and biochemistry are described. Particular attention is given to stereochemical considerations. Biosynthetic pathways for podophyllotoxin and congeners are surveyed. The binding to tubulin and the effects on microtubule assembly and disassembly are described and compared. In addition, structural features important to binding are examined using available analogs. Several features significant for tubulin interaction are common to these compounds and to colchicine. These are described and the implications for tubulin structure are discussed. The manifold results of applying these agents to biological systems are reviewed. These actions include effects that are clearly microtubule mediated and others in which the microtubule role is less obvious. Activity of some of these compounds due to inhibition of DNA topoisomerase is discussed. The range of species in which these compounds occur is examined and in the case of podophyllotoxin is found to be quite broad. In addition, the range of species that are sensitive to the effects of these compounds is discussed.

When was a "negative" clinical trial big enough? How many patients you needed depends on what you found.

"Negative" clinical trials that conclude that neither of the treatments is superior are often criticized for having enrolled too few patients. These criticisms usually are based on formal sample size calculations that compute the numbers of patients required prospectively, as if the trial had not yet been carried out. We suggest that this "prospective" sample size calculation is incorrect, for once the trial is over we have "hard" data from which to estimate the actual size of the treatment effect. We can either generate confidence limits around the observed treatment effect or retrospectively compare it with the effect hypothesized before the trial. If the observed effect is small, the risk of the false-negative conclusion (and the sample size required to draw negative or equivalency conclusions) is often much less than that generated by the "prospective" calculation.

Heroin vs morphine for cancer pain?

Narcotic analgesics are the mainstay of pain control in patients with cancer. A controversy has been raging in the United States and Canada as to the legalization of heroin. We have reviewed the literature in order to determine the relative efficacy of heroin and morphine in cancer pain. We applied the following methodologic criteria: Was the assignment of patients to the different opiates randomized? Were all clinically relevant outcomes reported? Were the patients recognizable? Were both clinical and statistical significance considered? Was the opiate regimen feasible in routine clinical practice? Were all patients who entered the study accounted for at its conclusion? Two trials satisfied our first standard. The first, a double-blind cross-over trial, failed to meet standard 4 (the negative conclusion may represent a type 2 error) and only 21% of patients completed both treatment periods. The second study, which compared intramuscular heroin and morphine among patients with postoperative pain, failed to meet standards 3 (patients not described in sufficient detail and only tangentially related to chronic cancer pain) and 4 (type 2 error). Thus the relative efficacy of heroin and morphine in the relief of chronic cancer pain remains unknown. Randomized trials that meet all six methodologic standards must therefore be carried out for this controversy to be resolved.

Problems in the handling of clinical and research evidence by medical practitioners.

There are important problems in the accuracy with which we collect, interpret, communicate, and apply clinical and relevant research evidence in the care of our patients. Many of these problems can be avoided or ameliorated by applying some specific measurement principles and information tools. The collection of clinical evidence can be improved by adhering to strategies that reduce observer error. The interpretation of clinical and paraclinical information can be improved by harnessing the predictive value of this information to estimates of the diagnosis, prognosis, and therapeutic responsiveness of patients. Communication can be improved by replacing the ambiguous argot of clinical equivocation with a more precise terminology. The detection of both valid and useful new knowledge can be facilitated by applying some straightforward guidelines to the rapid critical appraisal of the medical literature. Finally, we can look to advances in information technology to help us become more consistent in providing the best possible care for our patients.

Pulmonary embolism in outpatients with pleuritic chest pain.

Pleuritic chest pain is a frequent complaint in patients coming to the emergency room, but the proportion of such patients with pulmonary embolism is uncertain. In a prospective study, we evaluated the diagnostic outcomes in 173 consecutive patients who came to the emergency room with pleuritic chest pain. Pulmonary embolism, as demonstrated by angiography or autopsy, was present in 36 (21%). The need for objective testing is clearly indicated by our finding that the sensitivity (85%) and specificity (37%) of predetermined clinical variables for pulmonary embolism were insufficient to allow a definitive treatment decision. Optimal sensitivity and specificity are obtained by using pulmonary angiography in combination with lung scanning. The proportion of patients requiring angiography is substantially reduced, from 43% to 26%, without significant loss of accuracy, if ventilation imaging and impedance plethysmography are used together with perfusion scanning.

Cation selective promotion of tubulin polymerization by alkali metal chlorides.

A role for charge-based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher-order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+ > K+ > Li+ > Cs+, with optimal concentrations for Na+ at approximately 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of approximately 2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from approximately 0.9 to 0.99, and reverse or prevent urea-induced depolymerization of microtubules. In the presence of microtubule-associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selective persists. Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate-rich C termini of the tubulin monomers.

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

Intermediate filaments and steroidogenesis in adrenal Y-1 cells: acrylamide stimulation of steroid production.

The possible role of intermediate filaments in steroidogenesis was investigated in Y-1 mouse adrenal tumor cells by treatment with acrylamide, which is thought to disrupt intermediate filaments without directly affecting microtubules or microfilaments. Treatment of cells with 5 mM acrylamide increases steroidogenesis after a lag period of 4-6 h and induces rounding of the cells at approximately the same time. The effect of acrylamide on steroidogenesis is not cAMP mediated and occurs before pregnenolone formation. DNA synthesis is inhibited, while protein synthesis is not. Acrylamide does not affect polymerization/depolymerization of microtubules in vitro. Acrylamide stimulation of steroidogenesis is additive with that produced by either colchicine or ACTH, implying that acrylamide, ACTH, and colchicine act at different rate-limiting steps in steroidogenesis. In addition, acrylamide stimulation is additive with that of forskolin. Pretreatment of cells with taxol, an agent that specifically promotes microtubule polymerization, decreases acrylamide-stimulated (as well as colchicine or ACTH-stimulated) steroidogenesis, implying that there must also be some shared elements in the stimulating pathways. We hypothesize that regulation of steroidogenesis in the Y-1 cell depends on 1) disruption of a vimentin or tubulin coat surrounding lipid droplets and 2) possible functional shortening of the distance between cholesterol droplets and the mitochondrion. However, because of interactions between cytoplasmic fibers, it is currently impossible to say whether interruption of any one of them is a direct or indirect stimulus of steroidogenesis.

Can simple clinical measurements detect patient noncompliance?

Measurement of patient compliance is essential if management of low compliance is to be performed efficiently. We assessed the value of several easily obtained clinical assessments compared to quantitative pill counts among 134 newly treated hypertensive male steelworkers during the first 6 months of their treatment with antihypertensive medication. Patient's self-reports obtained on structured interview correlated best with pill count compliance (r = 0.74, p less than 0.0001). Patients overestimated their compliance by an average of 17% but 90% of those who admitted to being noncompliant were found so. Qualitative urinary chlorthalidone and hydrochlorothiazide levels and changes in serum potassium, uric acid, and blood pressure also correlated with pill count compliance but were less accurate than interviews. Assessment of the patient's "health beliefs" and a variety of sociodemographic and health traits and perceptions did not provide useful information on compliance. Interviewing the patient is a simple and useful approach in assessing compliance with antihypertensive therapy.

The multiple origins of cooperativity in binding to multi-site lattices.

Binding events involving the reversible association of ligands with polymeric lattices of binding sites are common in biology and frequently exhibit significant cooperativity in binding. Positive and negative cooperativity in binding may be detected by characteristic changes in binding curves for multiple binding, compared to the binding expected for simple, independent binding events that are based on combinatorial considerations only. Cooperativity arises from ligand-dependent interactions distinct from binding per se. Ligand-dependent nearest neighbor interactions may be of two types referred to as ligand-lattice (which can only occur if a bound ligand is unneighbored) and ligand-ligand (which can occur if two or more bound ligands are adjacent). The molecular mechanisms underlying these two sources of cooperativity are not the same. Identical cooperative binding curves may be produced by changes from unity in parameters representing either one or both of these interaction types. Positive cooperativity may equally result from destabilizing ligand-lattice interactions that disfavor initial, unneighbored binding, stabilizing ligand-ligand interactions that favor subsequent, neighbored binding, or both. The structural origins of these are different, and cooperativity may emerge from multiple structural interactions.

Human interferon-alpha receptor: identification of the region involved in binding to interferon-alpha B.

Three polypeptides comprising amino acids 1-102, 93-260, and 261-410 of the extracellular domain of the human interferon-alpha receptor HuIFN-alpha R (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990; 60:225-234) have been expressed in Escherichia coli. The polypeptides were sequestered within bacterial inclusion bodies. Inclusion body material was solubilized by 8 M urea, and the polypeptides were purified by gel filtration or histidine tag-based affinity chromatography. Overall recovery of each purified and refolded polypeptide was approximately 0.5-0.8 mg/liter of cell culture. The polypeptides migrated as homogeneous monomers of 12 kDa, 22 kDa, and 17 kDa, respectively on reduced sodium dodecylsulfate polyacrylamide gel electrophoresis. The polypeptide fragments corresponding to amino acids 1-102, and 93-260 of the extracellular domain of HuIFN-alpha R lacked the ability to bind to IFN-alpha B and to inhibit its biologic activities. The polypeptide fragment corresponding to amino acids 261-410 of the receptor molecule inhibited the antiproliferative activity of IFN-alpha B and competed with the Daudi cell surface receptor for binding to this IFN-alpha species.

Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli.

The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.

Active-control equivalence trials and antihypertensive agents.

PURPOSE: To identify methodological features that affect the validity of conclusions drawn from active-control equivalence trials and to apply these criteria to recently published trials comparing antihypertensive agents from different classes. METHODS: Standard methodological criteria for randomized clinical trials and six additional methodological features that affect the validity of active-control equivalence trials were applied to four recently published large trials that compared different antihypertensive classes and that concluded that their results showed equivalence. RESULTS: All four of these trials fulfilled standard criteria for randomized trials. However, none fulfilled all of the six additional methodological criteria that affect the validity of active-control equivalence trials, one fulfilled five criteria, two fulfilled two criteria, and one failed to fulfill any of the criteria. CONCLUSION: Standard methodological criteria for evaluating superiority trials are inadequate for the interpretation of active-control equivalence trials. The methodological criteria outlined in this article for judging the validity of active-control equivalence trials are not specific to antihypertensive trials and may be applied to trials that test a wide variety of interventions.

Purification, characterization, and drug susceptibility of tubulin from Leishmania.

Past work suggests that tubulin from kinetoplastid parasites may present an excellent drug target. To explore this possibility, tubulin was purified on a milligram scale from Leishmania mexicana amazonensis promastigotes by sonication, DEAE-Sepharose chromatography, and one cycle of assembly-disassembly. Purified leishmanial tubulin is recognized by commercially available anti-tubulin antibodies and displays concentration dependent assembly in vitro. The vinca site agents vinblastine, maytansine, and rhizoxin bind to leishmanial tubulin as assessed by the quenching of intrinsic tubulin fluorescence and the alteration of the proteins reactivity with the sulfhydryl-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid). They also interfere with the assembly of leishmanial tubulin at low micromolar concentrations. Electrophilic compounds such as phenyl arsenoxide and 4-chloro-3,5-dinitro-alpha,alpha,alpha-trifluorotoluene (chloralin), which are of interest as traditional and experimental antiparasitic agents, respectively, inhibit the assembly of leishmanial tubulin in vitro as well. Colchicine-site agents and trifluralin, on the other hand, have little or no effect on leishmanial tubulin in these assays. Maytansine, taxol, and the electrophiles block the growth of Leishmania donovani amastigote-like forms in vitro at low ( <1 microM) concentrations, while colchicine site agents, trifluralin, vinblastine, and rhizoxin are at least two orders of magnitude less toxic to the parasite.

Failure of weight reduction to reduce mildly elevated blood pressure: a randomized trial.

To determine the value of weight reduction on blood pressure, we randomly allocated 60 untreated, mildly hypertensive, obese individuals to a no-treatment control group or to a behaviourly-oriented weight loss ('diet') programme administered by professional dietitians. Behavioural techniques included self-monitoring, shaping, reinforcement and modelling. Subjects were reassessed after six months by an observer who was unaware of their study group. Fifty-four subjects (90%) completed the study. Diet subjects lost 4.1 kg and controls only 0.8 kg (P = 0.018). However, neither systolic nor diastolic blood pressures differed. The chance that we missed a clinically important diastolic difference of 6 mmHg (our pre-study target) is less than 1%. We conclude that our weight loss programme was successful in reducing weight but that weight loss is not useful in lowering blood pressure in mild, otherwise untreated hypertensives.

Hypertension control in two Canadian communities: evidence for better treatment and overlabelling.

We evaluated the prevalence and control of hypertension in two Canadian cities without university medical centre facilities. A stratified multistage probability sample was selected, and we interviewed 6258 adults between the ages of 30 and 69 inclusive. Blood pressure measurements were obtained during home interviews. Up to two further visits were made to people with untreated blood pressure elevation. By a diagnostic criterion of 90 mmHg, the hypertension prevalence was 114/1000. Six per cent of the hypertensives were undetected, 6% detected but untreated, 17% treated but uncontrolled and 70% were being treated and controlled. Control was better in females and older subjects. These findings show no disadvantages to hypertensives living away from university medical centres. We found a hypertension prevalence of 143/1000 among people who reported being diagnosed as hypertensive but who had normal blood pressure while not on medication. These results suggest a problem with over-labelling of hypertensives.