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1.  In vivo efficacy of enabling formulations based on hydroxypropyl-β-cyclodextrins, micellar preparation and liposomes for the lipophilic cannabinoid CB2 agonist, MDA7. 
Journal of pharmaceutical sciences  2012;102(2):352-364.
Enabling formulation based on hydroxypropyl-β-cyclodextrins (HPβCD), micellar preparation and liposomes have been designed to deliver the racemic mixture of a lipophilic CB2 agonist, MDA7. The antiallodynic effects of MDA7 formulated in these three different systems were compared after intravenous administration in rats. Stoichiometry of the inclusion complex formed by MDA7 in HPβCD was determined by continuous variation plot, ESI-MS analysis, phase solubility and NMR studies and indicate formation of exclusively 1:1 adduct. Morphology and particle sizes determined by DLS and TEM show the presence of a homogenous population of closed round-shaped oligolamellar MDA7 containing-liposomes, with average size of 117nm [polydispersity index (PDI) <0.1]. Monodisperse micelles exhibited average size of 15 nm (PDI 0.1). HPβCD based formulation administrated in vivo was composed of two discrete particles populations with a narrow size distribution of 3 nm (PDI<0.1) and 510 nm (PDI<0.1). HPβCD based formulation dramatically improved antiallodynic effect of MDA7 in comparison to the liposomes preparation. Through inclusion complexation and possibly formation of aggregates, HPβCD can enhance the aqueous solubility of lipophilic drugs thereby improving their bioavailability for i.v. administration.
doi:10.1002/jps.23393
PMCID: PMC3981967  PMID: 23192786
Chirality; Complexation; Cyclodextrins; Micelle; Bioavailability; CNS; Liposomes
2.  Microbial Carriage State of Peripheral Blood Dendritic cells (DCs) in Chronic Periodontitis Influences DC Differentiation, Atherogenic Potential† 
The low grade oral infection chronic periodontitis (CP) has been implicated in coronary artery disease risk, but the mechanisms are unclear. Here, a pathophysiological role for blood dendritic cells (DCs) in systemic dissemination of oral mucosal pathogens to atherosclerotic plaques was investigated in humans. The frequency and microbiome of CD19−BDCA-1+DC-SIGN+ blood myeloid DCs (mDCs) were analyzed in CP subjects with, or without existing acute coronary syndrome (ACS) and in healthy controls (CTL). FACS analysis revealed a significant increase in blood mDCs in the following order: CTL
doi:10.4049/jimmunol.1201053
PMCID: PMC3459682  PMID: 22891282
blood dendritic cells; Porphyromonas gingivalis; atherosclerosis; chronic periodontitis; acute coronary syndrome
Background
The Institute of Medicine (IOM) has called for a new health care paradigm that integrates patient values into discussions of the risks and benefits of treatment. Although CVD affects one-third of Americans, little is known about how adults regard the potential harms or complications of treatment.
Objective
We sought to determine the preferences of community dwelling adults for 15 potential harms or complications resulting from treatment of cardiovascular disease (CVD).
Methods
In a telephone survey, adults over 18 years of age residing on Long Island, New York were asked to score the preferences for 15 potential harms or complications of treatment of CVD on a scale from 0 to 100. All statistical analyses were based on non-parametric methods. Multivariable general linear model analyses were performed to identify demographic factors associated with the score assigned for each adverse outcome.
Results
The 807 individuals surveyed generated 723 unique sequences of scores for the 15 outcomes. The ranking of scores from least to most acceptable was stroke, major myocardial infarction (MI), cognitive dysfunction, renal failure, death, prolonged ventilator support, heart failure, angina, sternal wound infection, major bleeding, re-operation, prolonged recovery in a nursing home, cardiac readmission, minor MI and percutaneous coronary intervention. Demographic factors accounted for less than 7% of the observed variation in the score attributed to each outcome.
Conclusions
Individual community-dwelling adults living on Long Island, New York assign unique values to their preferences for potential harms encountered following treatment of CVD. Thus, risk-benefit discussions and treatment decisions regarding CVD should be harmonized to the value system of each individual.
doi:10.1177/0272989X13475717
PMCID: PMC3749300  PMID: 23407665
Anesthesia and Analgesia  2012;114(5):1104-1120.
Background
Peripheral neuropathy is a major dose-limiting toxicity of chemotherapy, especially after multiple courses of paclitaxel. The development of paclitaxel-induced neuropathy is associated with the activation of microglia followed by the activation and proliferation of astrocytes, and the expression and release of proinflammatory cytokines in the spinal dorsal horn. Cannabinoid type 2 (CB2) receptors are expressed in the microglia in neurodegenerative disease models.
Methods
To explore the potential of CB2 agonists for preventing paclitaxel-induced neuropathy, we designed and synthesized a novel CB2-selective agonist, namely MDA7. The effect of MDA7 in preventing paclitaxel-induced allodynia was assessed in rats and in CB2+/+ and CB2–/– mice. We hypothesize that the CB2 receptor functions in a negative-feedback loop and that early MDA7 administration can blunt the neuroinflammatory response to paclitaxel and prevent mechanical allodynia through interference with specific signaling pathways.
Results
We found that MDA7 prevents paclitaxel-induced mechanical allodynia in rats and mice in a dose- and time-dependent manner without compromising paclitaxel's antineoplastic effect. MDA7's neuroprotective effect was absent in CB2-/- mice and was blocked by CB2 antagonists, suggesting that MDA7's action directly involves CB2 receptor activation. MDA7 treatment was found to interfere with early events in the paclitaxel-induced neuroinflammatory response as evidenced by relatively reduced Toll-like receptor and CB2 expression in the lumbar spinal cord, reduced levels of extracellular signal regulated kinase 1/2 activity, reduced numbers of activated microglia and astrocytes, and reduced secretion of proinflammatory mediators in vivo and in in vitro models.
Conclusions
Our findings suggest an innovative therapeutic approach to prevent chemotherapy-induced neuropathy and may permit more aggressive use of active chemotherapeutic regimens with reduced long-term sequelae.
doi:10.1213/ANE.0b013e31824b0191
PMCID: PMC3334436  PMID: 22392969
Bacillus anthracis, the bacterium that causes anthrax, is an agent of bioterrorism. The most effective antimicrobial therapy for B. anthracis infections is unknown. An in vitro pharmacodynamic model of B. anthracis was used to compare the efficacies of simulated clinically prescribed regimens of moxifloxacin, linezolid, and meropenem with the “gold standards,” doxycycline and ciprofloxacin. Treatment outcomes for isogenic spore-forming and non-spore-forming strains of B. anthracis were compared. Against spore-forming B. anthracis, ciprofloxacin, moxifloxacin, linezolid, and meropenem reduced the B. anthracis population by 4 log10 CFU/ml over 10 days. Doxycycline reduced the population of this B. anthracis strain by 5 log10 CFU/ml (analysis of variance [ANOVA] P = 0.01 versus other drugs). Against an isogenic non-spore-forming strain, meropenem killed the vegetative B. anthracis the fastest, followed by moxifloxacin and ciprofloxacin and then doxycycline. Linezolid offered the lowest bacterial kill rate. Heat shock studies using the spore-producing B. anthracis strain showed that with moxifloxacin, ciprofloxacin, and meropenem therapies the total population was mostly spores, while the population was primarily vegetative bacteria with linezolid and doxycycline therapies. Spores have a profound impact on the rate and extent of killing of B. anthracis. Against spore-forming B. anthracis, the five antibiotics killed the total (spore and vegetative) bacterial population at similar rates (within 1 log10 CFU/ml of each other). However, bactericidal antibiotics killed vegetative B. anthracis faster than bacteriostatic drugs. Since only vegetative-phase B. anthracis produces the toxins that may kill the infected host, the rate and mechanism of killing of an antibiotic may determine its overall in vivo efficacy. Further studies are needed to examine this important observation.
doi:10.1128/AAC.01109-10
PMCID: PMC3294912  PMID: 22155821
Heart International  2009;4(1):e9.
The impact of left ventricular hypertrophy (LVH) on survival among patients with established coronary artery disease (CAD) is not well understood. We sought to evaluate the effect of LVH on the survival of patients with CAD following percutaneous coronary intervention (PCI). Three hospitals in New York City contributed prospectively defined data on 4284 consecutive patients undergoing PCI. Allcause mortality at a mean follow-up of three years was the primary endpoint. LVH was present in 383 patients (8.9%). LVH patients had a greater prevalence of hypertension (88% vs. 68%, p<0.001), vascular disease (21% vs. 6.6%, p=0.001), and prior heart failure (10% vs. 5.5%, p<0.001). LVH patients presented less often with one-vessel disease (38% vs. 50%, p=0.040) and more often with two- (34% vs. 29%, p=0.014) or three-vessel (22% vs. 18%, p=0.044) disease. Ejection fractions and angiographic success were similar in both groups. In-hospital mortality did not differ between groups. At three-year follow-up, the survival rate for patients with LVH was 86% vs. 91% in patients without LVH (log-rank p=0.001). However, after adjustment for differences in baseline characteristics using Cox proportional hazards analysis, LVH was found not to be an independent predictor of mortality (hazard ratio, 0.93; 95% confidence interval, 0.68–1.28; p=0.67). We conclude that LVH at the time of PCI is not independently associated with an increase in the hazard of death at three years.
doi:10.4081/hi.2009.e9
PMCID: PMC3184696  PMID: 21977285
angioplasty; stent; left ventricular hypertrophy; survival.
Bacillus anthracis causes anthrax. Ciprofloxacin is a gold standard for the treatment of anthrax. Previously, using the non-toxin-producing ΔSterne strain of B. anthracis, we demonstrated that linezolid was equivalent to ciprofloxacin for reducing the total (vegetative and spore) bacterial population. With ciprofloxacin therapy, the total population consisted of spores. With linezolid therapy, the population consisted primarily of vegetative bacteria. Linezolid is a protein synthesis inhibitor, while ciprofloxacin is not. Since toxins are produced only by vegetative B. anthracis, the effect of linezolid and ciprofloxacin on toxin production is of interest. The effect of simulated clinical regimens of ciprofloxacin and linezolid on the vegetative and spore populations and on toxin production was examined in an in vitro pharmacodynamic model over 15 days by using the toxin-producing Sterne strain of B. anthracis. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. With ciprofloxacin therapy, the total Sterne population consisted of spores. With linezolid therapy, >90% of the population was vegetative B. anthracis. With ciprofloxacin therapy, toxin was first detectable at 3 h and remained detectable for at least 5 h. Toxin was never detected with linezolid therapy. Ciprofloxacin and linezolid reduced the total Sterne population at similar rates. However, the B. anthracis population was primarily spores with ciprofloxacin therapy and was primarily vegetative bacteria with linezolid therapy. Toxin production was detected for at least 5 h with ciprofloxacin therapy but was never detected with linezolid treatment. Linezolid may have an advantage over ciprofloxacin for the treatment of B. anthracis infections.
doi:10.1128/AAC.05724-11
PMCID: PMC3256020  PMID: 22064542
Dermatobia hominis, the botfly, is indigenous to Central and South America. Its usual host is a mammal, often a horse or cow. Cutaneous furuncular myiasis, human infestation by the botfly, has rarely been reported. Symptoms of infestation include a locally painful, firm furuncular lesion, often with a centrally located pore. Due to their infrequent occurrence, these lesions are often misdiagnosed as cellulitis, leishmaniasis, furunculosis, staphylococcal boil, insect bite or sebaceous cyst – conditions with similar presentations. The present case reiterates the need to think of ‘zebras’ when hearing ‘hoof beats’ that may have originated in a different land.
PMCID: PMC2539025  PMID: 19554228
Botfly; Furuncular myiasis; Sebaceous cyst
Environmental Health  2011;10:90.
Background
Dried meat and fish have served as an important durable nutrition source for humans for centuries. Because omega 3 fatty acids in fish are recognized as having antioxidant and anti inflammatory properties found to be beneficial for good health, many consumers are looking to fish as their main source of protein. Unfortunately, contaminants such as methylmercury can accumulate in some species of fish. The purpose of this research is to test commercially available fish jerky snack foods for mercury contamination.
Methods
Fifteen bags of marlin jerky, three bags of ahi jerky, and three bags of salmon jerky were purchased from large retail stores in Hawaii and California, and directly from the proprietors' Internet websites. Five individual strips of jerky per bag were analyzed for a total of one hundred and five tests.
Results
From the seventy-five marlin jerky samples, mercury concentration ranged from 0.052-28.17 μg/g, with an average of 5.53 μg/g, median 4.1 μg/g. Fifty-six (75%) marlin samples had mercury concentrations that exceeded the FDA's current mercury action level of 1.0 μg/g, while six samples had greater than 10 μg/g. Fifteen samples of ahi had mercury concentrations ranging from 0.09-0.55 μg/g, while mercury concentrations in fifteen salmon samples ranged from 0.030-0.17 μg/g.
Conclusions
This study found that mercury concentrations in some fish jerky can often exceed the FDA's allowable mercury limit and could be a significant source of mercury exposure.
doi:10.1186/1476-069X-10-90
PMCID: PMC3206828  PMID: 21988746
Ahi; fish; jerky; marlin; mercury; methylmercury; salmon
BMJ Open  2011;1(2):e000137.
Introduction
Corticosteroid injections (CSI) are a recommended and often-used first-line intervention for shoulder impingement syndrome (SIS) in primary care and orthopaedic settings. Manual physical therapy (MPT) offers a non-invasive approach with negligible risk for managing SIS. There is limited evidence to suggest significant long-term improvements in pain, strength and disability with the use of MPT, and there are conflicting reports from systematic reviews that question the long-term efficacy of CSI. Specifically, the primary objective is to compare the effect of CSI and MPT on pain and disability in subjects with SIS at 12 months.
Design
This pragmatic randomised clinical trial will be a mixed-model 2×5 factorial design. The independent variables are treatment (MPT and CSI) and time with five levels from baseline to 1 year. The primary dependent variable is the Shoulder Pain and Disability Index, and the secondary outcome measures are the Global Rating of Change and the Numeric Pain Rating Scale. For each ANOVA, the hypothesis of interest will be the two-way group-by-time interaction.
Methods and analysis
The authors plan to recruit 104 participants meeting established impingement criteria. Following examination and enrolment, eligible participants will be randomly allocated to receive a pragmatic approach of either CSI or MPT. The MPT intervention will consist of six sessions, and the CSI intervention will consist of one to three sessions. All subjects will continue to receive usual care. Subjects will be followed for 12 months.
Dissemination and ethics
The protocol was approved by the Madigan Army Medical Center Institutional Review Board. The results may have an impact on clinical practice guidelines. This study was funded in part by the Orthopaedic Physical Therapy Products Grant through the American Academy of Orthopaedic Manual Physical Therapists.
Trial Registration
http://clinicaltrials.gov/ NCT01190891.
Article summary
Article focus
Shoulder pain is a common symptom in patients seeking healthcare for musculoskeletal complaints.
Corticosteroid injections (CSI) are a common first-line intervention for shoulder pain in primary care settings, but their long-term efficacy has not been established.
The long-term efficacy of manual physical therapy and CSI will be evaluated and compared from baseline out to 1 year after enrollment.
Key messages
Manual physical therapy has been shown to provide improvements in pain and function in patients with shoulder impingement but has not been directly compared with CSI.
Understanding which interventions have better long-term outcomes may be instrumental in helping improve clinical practice guidelines for the management of shoulder impingement syndrome.
Strengths and limitations of this study
This randomised controlled study will compare the effectiveness of a manual physical therapy approach to a corticosteroid injection in patients with shoulder impingement.
This is a pragmatic study evaluating two interventions that are standard practice and have been shown to be effective for shoulder impingement.
Even as a single blinded randomised clinical trial there is no true control group, and we cannot state whether true a cause-and-effect relationship exists.
Owing to the pragmatic nature of the study, the intervention will not be standardised, which could make it difficult for clinicians to replicate.
The lack of a gold standard with the diagnosis of shoulder impingement makes this population difficult to study.
doi:10.1136/bmjopen-2011-000137
PMCID: PMC3191586  PMID: 22021870
New approaches are needed for the treatment of Pseudomonas aeruginosa infections. All available single agents are suboptimal, especially for resistance suppression. Classical β-lactam/aminoglycoside combinations are not used often enough at least in part because of concern for nephrotoxicity. We evaluated the combination of meropenem and levofloxacin against the P. aeruginosa PAO1 wild type and its isogenic MexAB pump-overexpressed mutant. The drugs were studied using an in vitro hollow-fiber pharmacodynamic infection model. There were 16 different regimens evaluated for both isolates. Both total population and resistant subpopulations were quantified. Drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The impact of monotherapy versus that of combination therapy for attainment of a 3-log cell kill and for resistance suppression was examined using Kaplan-Meier analysis. Drug exposures were calculated by fitting the concentration-time data using the ADAPT II package of programs. For both isolates, monotherapy allowed resistance emergence with all but the largest exposure or with all exposures. In contrast, there was no resistance emergence with any combination regimen. Kaplan-Meier analysis showed significant differences in time to attainment of a 3-log cell kill as well as time to resistance emergence for monotherapy and combination therapy for both isolates, in favor of the combination regimens. Determination of the pharmacodynamic indices associated with resistance suppression demonstrated a 2- to 3-fold reduction with the use of combinations. Combination therapy with meropenem and levofloxacin provides a significantly faster time to attain a 3-log cell kill and significantly better resistance suppression than does either monotherapy. This combination should be evaluated in a clinical trial.
doi:10.1128/AAC.00065-10
PMCID: PMC2876409  PMID: 20368395
mBio  2010;1(3):e00139-10.
Moxifloxacin is under development for expanded use against Mycobacterium tuberculosis. Rifampin is a mainstay of therapy. We examined the interaction of moxifloxacin plus rifampin for log-phase and nonreplicating persister (NRP) organisms. For this evaluation, we employed our hollow-fiber infection model, in which organisms are exposed to clinically relevant drug concentration-time profiles and the impact on bacterial cell kill and resistant subpopulation amplification is determined. In log phase, resistance emergence was observed in all monotherapy regimens and in no combination therapy regimen. No difference was seen in time to a 3-log reduction in the bacterial burden; there was a significant difference in time to resistance emergence (P = 0.0006). In the NRP experiment, no resistance emergence was seen. There was a significant difference between the monotherapy and combination therapy regimens in time to a 3-log reduction in the bacterial burden (P = 0.042). The combination is efficacious for suppressing resistant organisms but is antagonistic for cell kill.
IMPORTANCE
M. tuberculosis infects one-third of the world’s population. Multiresistant organisms have become more frequent, threatening our ability to provide adequate chemotherapy. Moxifloxacin has been seen as an important new agent with the potential to supplant isoniazid or add to the rifampin/isoniazid combination. M. tuberculosis also exists in different physiological states, including the nonreplicating persister phenotype. We examined the moxifloxacin/rifampin combination in a new in vitro system to allow judgment of how moxifloxacin would interact with rifampin and allow its performance in clinical trials to be placed into perspective. Importantly, the combination suppressed resistance emergence, but at the price of slightly slowing bacterial cell kill. This new combination is a welcome addition to the physician’s armamentarium.
doi:10.1128/mBio.00139-10
PMCID: PMC2925073  PMID: 20802826
Background
Despite compelling evidence that aspirin reduces fatal and non-fatal vascular events among the overall population in various settings, women have frequently been underrepresented and their data underreported. We sought to evaluate the relationship between aspirin use, dose (81 or 325mg) and clinical outcomes among postmenopausal women with stable cardiovascular disease.
Methods
Women with cardiovascular disease (n=8928) enrolled in the Women’s Health Initiative Observational Study were used for this analysis. The primary outcome was the incidence of all-cause mortality and cardiovascular events (myocardial infarction, stroke and cardiovascular death).
Results
Among 8928 women with stable cardiovascular disease, 4101 (46%) reported taking aspirin, of whom 30% were on 81 and 70% were on 325mg. At 6.5 years of follow-up, no significant association was noted for aspirin use and all-cause mortality or cardiovascular events. However, after multivariate adjustment, aspirin use was associated with a significantly lower all-cause (adjusted HR 0.86, [0.75-0.99], P=0.04) and cardiovascular related mortality (adjusted HR 0.75, [0.60-0.95], P=0.01) compared with no aspirin. Aspirin use was associated with a lower risk of cardiovascular events (adjusted HR 0.90, [0.78-1.04], P=0.14) which did not meet statistical significance. Compared with 325mg, use of 81mg was not significantly different for all-cause mortality, cardiovascular events or any individual endpoint.
Conclusions
After multivariate adjustment, aspirin use was associated with significantly lower risk of all-cause mortality, specifically cardiovascular mortality, among postmenopausal women with stable cardiovascular disease. No significant difference was noted between 81 and 325mg of aspirin. Overall, aspirin use was low in this cohort of women with stable cardiovascular disease.
doi:10.1161/CIRCOUTCOMES.108.791269
PMCID: PMC2801891  PMID: 20031819
Aspirin; Dose; Women; Cardiovascular Disease; Observational Study
Antimicrobial Agents and Chemotherapy  2007;51(11):3988-4000.
The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 μg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 μg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 μg/ml; the MICs of ciprofloxacin were 2 and 1 μg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin at 750 mg every 12 h also rapidly selected for ciprofloxacin-resistant mutants of Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the emergence of resistance. Levofloxacin at 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at the baseline, Spn-RC2 had greater EP expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of EP expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in EP expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged under suboptimal antibiotic regimens had a stable increase in EP expression. Levofloxacin at 500 mg QD in combination with reserpine, an EP inhibitor, or at 750 mg QD alone killed wild-type S. pneumoniae and strains that overexpressed reserpine-inhibitable EPs and was highly effective in preventing the emergence of fluoroquinolone resistance in S. pneumoniae during therapy. Ciprofloxacin at 750 mg every 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented the emergence of resistance in Spn-058 but not in Spn-RC2, the EP-overexpressing strain.
doi:10.1128/AAC.00391-07
PMCID: PMC2151412  PMID: 17846144
The Journal of Cell Biology  1974;61(2):514-536.
The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.
PMCID: PMC2109281  PMID: 4827911
The Journal of Cell Biology  1973;56(2):340-359.
In the first of two companion papers which attempt to correlate microtubules and their nucleating sites with developmental and cell division patterns in the unicellular flagellate, Ochromonas, the distribution of cytoplasmic and mitotic microtubules and various kinetosome-related fibers are detailed. Of the five kinetosome-related fibers, which have been found in Ochromonas, two, the kineto-beak fibers and the rhizoplast fibers are utilized as attachment sites for distinct groups of microtubules. The set of microtubules attached to the kineto-beak fibers apparently shape the anterior beak region of the cell whereas the rhizoplast microtubules appear to extend into and shape the tail in vegetative cells. In mitotic cells a rhizoplast is found at each spindle pole apparently serving as foci for the spindle microtubules. These findings are discussed in relation to the less well defined attachment sites for vegetative and mitotic microtubules in other kinds of cells. It is noted that the effects of depolymerizing microtubules in vivo might be easily quantitated in whole populations since no external wall or pellicle contributes to the maintenance or the biogenesis of the characteristic cell form of Ochromonas.
PMCID: PMC2108894  PMID: 4682900
The Journal of Cell Biology  1973;56(2):360-378.
The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.
PMCID: PMC2108910  PMID: 4682901

Results 1-17 (17)