Comparative study of ATR and transflection IR spectroscopic techniques for the analysis of hallucinogenic mushrooms.

This paper compares the use of ATR and transflection spectroscopic techniques for the qualitative analysis of psilocin extracted from hallucinogenic mushrooms and control spiked mushrooms. Both techniques gave comparable results and agreed with prior GC/MS analysis of the actual case samples.

Studying the effect of pH variation on the incorporation of the antipsychotic drug clozapine into dyed and non-dyed hair samples using micro-attenuated total reflection spectroscopy.

Antipsychotic drugs are among the mostly widely used medications and are usually taken for prolonged periods of time. Due to its accumulation and trapping of drugs, hair can provide a useful indication of long-term exposure. Of interest also is what if any changes in the structural components of hair occur as a result of the drug binding process. Micro-attenuated total reflection (ATR) spectroscopy is able to examine the structural changes of hair samples by the application of sufficient pressure and without microtoming the hair (A. Koçak and S. L. Berets, Appl. Spectrosc. 62, 803 (2008)). In this investigation, we examined changes resulting from exposure of dyed and undyed hair to external clozapine as a function of the pH of the exposing solution. Single samples from different individuals and in one case from different regions of the scalp from the same individual were analyzed. The results demonstrated that pH related differences exist between drug-exposed dyed and non-dyed samples.

Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic spindle checkpoint proteins at PtK1 kinetochores.

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle.

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.

Wegener's granulomatosis: role of environmental exposures.

OBJECTIVE: The etiology of Wegener's granulomatosis (WG) remains unknown. The predominant involvement of the airways and the presence of neutrophilic alveolitis at disease onset have led us to postulate that an inhaled agent may trigger the onset of WG. This study is designed to analyze differences in self-reported environmental exposures between patients with WG and various control populations. METHODS: We conducted a standard interviewer-administered questionnaire case controlled survey of 101 patients with WG, 54 healthy controls, 24 patients with sarcoidosis or idiopathic pulmonary fibrosis, and 45 patients with various inflammatory rheumatologic diseases. We assessed environmental exposures for one year prior to the onset of symptoms or prior to the interview date for healthy controls. RESULTS: Seasonal differences in the onset of WG were not apparent. More than 75% of the patients in all groups noted remarkable environmental exposure to inhaled substances (fumes or particulate matter), within one year prior to disease onset for WG and other diseases or prior to the interview date for healthy controls. Differences between WG and control groups were apparent in several categories of exposure. Statistically significant differences occurred in regard to a vocational exposure to fumes or particulate materials (WG > healthy controls and rheumatic disease controls), residential exposure to particulate materials from construction (WG > pulmonary disease controls) and occupational exposure to pesticides (WG > healthy, pulmonary and rheumatic disease controls). CONCLUSION: This study confirms the absence of seasonal differences in the onset of WG. It also demonstrates high rates of self-reported environmental exposures to inhaled substances in WG and all control populations. It is possible that more significant differences in the quality, quantity and intensity of exposure to inhaled potential precipitants of WG had occurred between groups, but were not detected by our survey. Alternatively, the absence of substantial differences in patients with WG and controls may reflect the more important role of host susceptibility factors.

Hemolytic complement activity in normal human donor corneas.

Normal human donor corneas were minced into small fragments and eluted for 16 to 23 hours at 4 degrees C. The corneal eluates were then studied for hemolytic complement activity of C1, C4, C2, C3, C5, C6, and C7 with 50% hemolysis (CH50) of sensitized sheep RBCs. Sera from ten normal volunteers were also assayed for hemolytic complement activity in CH50 units per milliliter. For each complement component, the mean hemolytic activity in corneas was compared with the mean hemolytic activity in sera. These comparisons suggest that molecular weight may be a factor in determining the concentration of complement components in the cornea. The present study provides normal values of hemolytic complement activity for further studies of complement consumption in corneal diseases.

Control of schistosomiasis: report of a workshop.

Nineteen scientists, field workers, and representatives of funding agencies active in schistosomiasis research and control met in Bellagio, Italy in October 1977 to attempt to evaluate the effectiveness of current control methods and what might be accomplished with available technology. The deliberations included summaries of knowledge on the biology, transmission, and control of schistosomiasis and assessment of major control programs and methodologies. The groups concluded that in the major endemic areas considerable gains in control of schistosomiasis could be made with current technology. However, maintenance of control in most countries, and establishment of serious control programs in countries in which schistosomiasis is a less severe public health problem, would require development of less expensive modalities which would need little monitoring and possibly have benefits extending beyond schistosomiasis control.

Behavioral community psychology: training a community board to problem solve.

This study demonstrated the effect of training nine lower socio-economic adults participating as policy board members in a federally funded rural community project to make behaviorally defined statements to increase problem-solving behaviors in board meetings. A multiple-baseline design across subjects and skills was used to analyze the behavioral categories of: (1) stating the problem; (2) finding solutions to the problem, and (3) implementing the action to the solution. Problem-solving responses during board meetings increased for subjects following training and remained higher than baseline during follow-up.