Active MAP kinase in mitosis: localization at kinetochores and association with the motor protein CENP-E.

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.

Development of liquid chromatographic method for simultaneous determination of mycophenolate mofetil and its degradation product mycophenolic acid in dosage form.

In this paper, a chemometrical approach is applied for the development of a reversed-phase high-performance liquid chromatography method for the simultaneous determination of mycophenolate mofetil and its degradation product mycophenolic acid in dosage form. The fractional factorial design is used in screening experiments for selecting the variables that significantly influence the chromatographic procedure. The investigated variables are column type, temperature of the column, and composition of the mobile phase (with respect to pH and the percentage of organic modifier). Investigation is performed using two columns, XTerra (RP 18, 150 mmx3.9 mm) and Chromolith (RP-18e, 100 mmx4.6 mm). Because the column type shows no influence on separation process, the Chromolith column is further used due to its ability to achieve a high-speed separation without loss of column efficiency. Total analysis time is reduced from 8.34 min on XTerra to 1.27 min on Chromolith. The columns' efficiency, analysis cost, and peak symmetries are briefly compared. For both substances, only two variables are found significant: percentage of acetonitrile and pH of the water phase. Afterward, the main variables are optimized using response surface methodology for visualization and easier identification of optimal conditions. The optimal conditions are obtained with mobile phase composition of acetonitrile-15 mM phosphate buffer (pH adjusted to 4.0 with 85% orthophosphoric acid) (35:65, v/v) at the flow rate of 5 mL/min. The temperature of the column is adjusted to 25 degrees C and detection is performed at 254 nm.

Investigation of chromatographic conditions for the separation of ofloxacin and its degradation products.

A sensitive, precise, and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous determination of fluoroquinolone antibacterial ofloxacin and its degradation products: decarboxy ofloxacin, 9-piperazino ofloxacin, des-methyl ofloxacin, and ofloxacin-N-oxide. The proposed RP-HPLC method allowed separation of all five compounds simultaneously with the total time of the analysis not more than 15 min. The relative standard deviation (RSD) values for quantification of DOF, POF, MOF, OF, and NOF were of 0.77, 0.58, 0.51, 0.10, and 0.70%, respectively, indicating a good precision of the method. The limits of detection for DOF, POF, MOF, OF, and NOF were 0.10, 0.13, 0.06, 0.03, and 0.03 microg mL(-1), respectively. The described method can be used for simultaneous identification and quantification of all analysed compounds in different pharmaceutical formulations.

Validation of a high-performance liquid chromatographic method for the simultaneous determination of tramadol and its impurities in oral drops as a pharmaceutical formulation.

The novel, rapid high performance liquid chromatographic method for the determination of tramadol hydrochloride and its three impurities was developed and validated. The method can simultaneously assay potassium sorbate, used as preservative, and saccharin sodium, used as sweetener in tramadol pharmaceutical formulation. The separation was carried out on a C(18) XTerra (150 mm x 4.6 mm, 5 mm) column using acetonitrile-0.015 M Na(2)HPO(4) buffer (2:8, v/v) as mobile phase (pH value 3.0 was adjusted with orthophosphoric acid) at a flow rate 1.0 ml min(-1), temperature of the column 20 degrees C and UV detection at 218 nm. The method was found to be linear (r > 0.999) in the range of 0.05-0.8 mg ml(-1) for tramadol hydrochloride, 0.1-1.2 mg ml(-1) for impurities B and C and for impurity A (r > 0.995) in the range 0.15-2.4 mg ml(-1). The low RSD values indicate good precision and high recovery values indicate excellent accuracy of the HPLC method. Developed method was successfully applied to the determination of tramadol hydrochloride, its investigated impurities and potassium sorbate in commercial formulation. The recovery of tramadol hydrochloride was 98.25% and RSD was 1.80%. The method is rapid and sensitive enough to be used to analyse Trodon oral drops.

Validation of liquid chromatographic method for analysis of lidocaine hydrochloride, dexamethasone acetate, calcium dobesilate, buthylhydroxyanisol and degradation product hydroquinone in suppositories and ointment.

In this paper, there was developed a sensitive, precise and accurate reversed-phase liquid chromatographic (RP-HPLC) method and validated for simultaneous determination of lidocaine hydrochloride, dexamethasone acetate (DA) and calcium dobesilate (CD) in suppositories and ointment. Also there was achieved a parallel analysis of buthylhydroxyanisol, as a preservative, and hydroquinone, as a degradation product of calcium dobesilate, present in these dosage forms. The relative standard deviation (RSD) values for all five compounds indicated a good precision and accuracy of the RP-HPLC method. Method is selective, sensitive and reproducible with good recovery values and can be applied in simultaneous determination of all mentioned compounds.

Validation of a high-performance liquid chromatography method for the determination of pancuronium in Pavulon injections.

A new high-performance liquid chromatography (HPLC) method was developed for the quality control of pancuronium bromide and its degradation products. The HPLC method used a 5-microm Supelcogel ODP-50 (150x4 cm) column with acetonitrile-CH3OH-water-F3CCOOH (20.5:74.9:0.1, v/v) as the mobile phase (pH value 2.0 adjusted with trifluoroacetic acid) at a flow-rate 0.8 ml/min and UV detection at 210 nm. The Beer's law plots were found to be linear over the concentration range 0.4-1.2 mg/ml of pancuronium bromide and 0.04-0.08 mg/ml of desacetyl degradation products (R2=0.9995). The RSD of the peak areas was 1.09% and the recovery was 102.43%. The RSD value shows good precision, acceptable accuracy and reproducibility of the new method for the determination of pancuronium bromide in presence of its desacetyl degradation products. The method is rapid and sensitive enough to be used for Pavulon injection analysis.

Use of ANN modelling in structure--retention relationships of diuretics in RP-HPLC.

Structure retention relationship study, conducted by RP HPLC, was used to investigate physical chemical parameters related to the RP retention times of amiloride, hydrochlorothiazide and methyldopa in order to predict the separation of amiloride and methylclothiazide from Lometazid tablets. Retention data were obtained with an ODS column using a mobile phase methanol water (pH adjusted with phosphoric acid). Physical chemical properties were calculated directly from the molecular structure. Artificial neural networks (ANNs) were used to correlate chromatograms retention times with mobile phase composition and pH, and with physical chemical properties of amiloride, hydrochlorothiazide and methyldopa and to predict separation of amiloride and methylclothiazide from Lometazid tablets. Sensitivity analysis was performed to interpret the meaning of the descriptors included in the models. Results confirmed the dominant role of the polar modifier in such chromatographic systems. Within a series of solutes chromatographed under identical conditions, the retention parameters could be approximated by a non-linear combination of logP, logD, pKa, surface tension, parachor, molar volume and to minor extend by polarisability, reetractivity index and density. This study has demonstrated that the use ANNs techniques can result in much more efficient use of experimental information. As HPLC is the most popular analytical technique, improvements in HPLC methods development can yield significant gains in the overall analytical effort. The ANNs extension presented could be the method of choice in some advanced research settings and serves as an indication of the broad potential of neural networks in chromatography analysis.

The use of a response surface methodology on HPLC analysis of methyldopa, amiloride and hydrochlorothiazide in tablets.

A multifactor optimisation technique is successfully applied to develop a new HPLC method in which methyldopa, hydrochlorothiazide and amiloride were analysed and determined on a C18 column with detection at 286 nm. The optimal conditions of HPLC separation were determined with the aid of the response surface diagram -- 'window diagram'. The effect of simultaneously varying the pH, proportion aqueous acetic acidum and methanol in the mobile phase were studied to optimise the separation. The mobile phase composition that provides an acceptable resolution methyldopa, hydrochlorothiazide and amiloride in a short elution time is water--methanol (75:25) and pH 3.60. The k' values for methyldopa, hydrochlorothiazide and amiloride after optimisation were 1.40, 2.50 and 5.33, respectively. Relative retention (alpha) for ratio hydrochlorothiazide/methyldopa and amiloride/hydrochlorothiazide were 1.767 and 2.159, respectively. Correlation coefficients of the calibration curves for all analytes were greater than 0.995 and the R.S.D. values for the slope and the intercept with respect to the linearity were less than 2%. A method is applied for the quantitative analysis of Alatan tablets (Lek-Ljubljana). The powdered tablets are extracted with methanol, containing caffeine as the internal standard and assayed by comparison of peak areas after liquid chromatography. The high recovery (for all analytes about 100%) and the low R.S.D. (<2%) confirm good precision and reproducibility of the chromatographic method.

Spectrophotometric study of diclofenac-Fe(III) complex.

A multifactor optimisation technique is successfully applied to develop a new spectrophotometric method in which diclofenac sodium is analysed and determined as it's Fe(III) complex. The effect of simultaneously varying the pH, ionic strength and concentration of colour reagents in the reaction mixture were studied. A four-variable two-level factorial design was used to investigate the significance of each variable and interactions between them. A response surface design was used to optimise complex formation and extraction. It was established that diclofenac reacts with Fe(III) chloride, in the presence of ammonium thiocyanate, in the pH range 4.2-6.5, forming a red chloroform extractable (2:1) complex with maximum absorbance at 481 nm. By applying the methods of Sommer and Job involving non-equimolar solutions the conditional stability constant of the complex, at the optimum pH of 6.0 and an ionic strength mu = 0.19M, was found to be 10(6.4). Good agreement with Beer's law was found for diclofenac concentrations up to mmol 1(-1). The nominal percent recovery of diclofenac was 98.8% (n = 10). The lower limit of sensitivity of the method was found to be 14.7 micrograms ml(-1).

Application of artificial neural networks in HPLC method development.

The use of artificial neural networks (ANNs) for response surface modelling in HPLC method development for amiloride and methychlothiazide separation is reported. The independent input variables were pH and methanol percentage in mobile phase. The outputs were capacity factors. The results were compared with a statistical method (multiple nonlinear regression analysis). Networks were able to predict the experimental responses more accurately than the regression analysis.

Statistical optimization of a reversed-phase liquid chromatographic method for the analysis of amiloride and hydrochlorothiazide in tablets.

A method has been developed for the separation of hydrochlorothiazide and amiloride by high-performance liquid chromatographic (HPLC) method on a C18 column with detection at 280 nm. The optimal conditions of separation were determined with the aid of 'window diagram' technique of Laub and Purnell. The effect of simultaneously varying the pH, proportion aqueous acetic acid and methanol in the mobile phase were studied to optimize the separation. A response surface diagram was used to optimize the experimental conditions for the separation. The mobile phase composition that provides an acceptable resolution hydrochlorothiazide and amiloride in a short elution time is water:methanol (60:40) and pH 3.2 (pH adjusted to 3.2 with CH3COOH). A method is applied for the quantitative analysis of Moduretic tablets (Merck Sharp & Dokme International). The powdered tablets are extracted with methanol, containing caffeine as the internal standard, and assayed by comparison of peak areas after liquid chromatography.

Application of experimental design to the development of an HPLC method for the analysis of ochratoxin A in Triticum aestivum grain.

Ochratoxin A is a mycotoxin, a natural product of Aspergillus and Penicillium species. It can be present in grain from Triticum aestivum, (Graminae) and other starch-abundant cereals. This paper describes the investigation of ochratoxin A in grain from Triticum aestivum using a statistically optimized HPLC method. The assay was developed using two mathematical statistical models: factorial design and response surface mapping. The final step was to optimize the values of variables by response surface design. The analysis of variance 'ANOVA' method was applied to the analytical results in order to construct an adequate model. The optimal experimental conditions obtained by the response surface diagram method were: pH = 2.5, composition of the mobile phase acetonitrile: water 55:45 v/v and flow rate 1.0 ml/min. with a C18 column. Retention time and capacity factor for ochratotoxin A were 7.46 min. and 1.19, respectively.

The effect of intramedullary corticocancellous bone plug for hip hemiarthroplasty.

BACKGROUND: A prospective review was performed on 60 consecutive patients with hip hemiarthroplasty after femoral neck fractures. METHODS: Twenty-two patients underwent Austin Moore hemiarthroplasty with an intramedullary corticocancellous bone plug at the tip of the prosthesis (group A) and 38 patients underwent Austin Moore hemiarthroplasty alone (group B). The patients were evaluated clinically and radiographically at 3 and 6 months postoperatively and annually thereafter. RESULTS: There was no statistically significant difference in thigh pain score between the two groups. At 3- and 6-month follow-up, 88% and 83% of group A patients experienced no pain or mild thigh pain, compared with 72% and 76% in group B, respectively. The radiographs revealed more stem subsidence and calcar osteolysis in group B than in group A (p < 0.01 and p < 0.05, respectively). Furthermore, in both groups there was a correlation between calcar atrophy, stem subsidence, and early clinical thigh pain score (p < 0.05). CONCLUSION: Our data suggest that, whereas the radiologic findings in both groups may be related to thigh pain, they had little effect on the rate of femoral stem revision. We believe that the application of a corticocancellous bone plug in uncemented hip hemiarthroplasty for treatment of femoral neck fractures can decrease the incidence of early thigh pain in the first 6 months.

Mediators of aldosterone action in the renal tubule.

The aldosterone-sensitive distal nephron extends from the second part of the distal convoluted tubule to the inner medullary collecting duct. As recently shown, aldosterone increases within two hours the abundance of the alpha-subunit of the epithelial sodium channel along the entire aldosterone-sensitive distal nephron, whereas it induces only in an initial portion of the aldosterone-sensitive distal nephron an apical translocation of all three epithelial sodium channel subunits. This suggests that another factor or factors determines the length of the aldosterone-sensitive distal nephron portion in which aldosterone controls epithelial sodium channel surface expression. Since the glucocorticoid-induced kinase SGK1 was identified as aldosterone-induced protein in 1999, it has been postulated to play a key regulatory role. The in-vivo localization of its induction to segment-specific cells of the aldosterone-sensitive distal nephron, and the in-vitro correlation of the amount of its hyperphosphorylated form with transepithelial sodium transport, support this hypothesis. Other recent studies unravel pathways other than those activated by aldosterone and insulin that impact on SGK1 expression and/or function, and thus shed some light onto the complex network that appears to control sodium transport. In view of the ongoing research, the question of how, and formally also whether, SGK1 acts on the epithelial sodium channel should be resolved in the near future.

Peripheral projections of a subpopulation of dorsal root ganglion neurons defined by ovalbumin immunoreactivity.

Previous studies from this laboratory have used antisera to aldehyde-conjugated ovalbumin to localize ovalbumin-like immunoreactivity within a subpopulation of sensory neurons. We have now combined retrograde tracing and immunohistochemical procedures to identify the tissues innervated by sensory neurons which are either immunoreactive or non-immunoreactive for ovalbumin. The fluorescent tracer Di-I was administered to feather follicles, flexor ulnar muscle, subdermis, expansor secundariorum, heart and liver and identified seven days later within corresponding dorsal root ganglia. Most neurons innervating the follicles had large cell somata, and fewer than 3% were immunoreactive for ovalbumin. In contrast, most sensory neurons projecting to subdermis, muscle and expansor secundariorum muscle were of a medium diameter. Approximately 25% of those neurons projecting to the expansor secundariorum, and 60% projecting to the subdermis and muscle, were immunoreactive for ovalbumin. Sensory neurons innervating heart and liver were the smallest, and only 8% were immunoreactive for ovalbumin. The study indicates that sensory neurons innervating different organs have somata with significantly different sizes, suggesting a functional specificity. Moreover, neurons demonstrating either the ovalbumin-IR positive or negative phenotypes show distinct peripheral projections, suggesting that this phenotype may be at least partially controlled by retrograde signals derived from the cells they innervate.

Pleiotropic action of aldosterone in epithelia mediated by transcription and post-transcription mechanisms.

The aldosterone-induced increase in sodium reabsorption across tight epithelia can be divided schematically into two functional phases: an early regulatory phase starting after a lag period of 20 to 60 minutes, during which the pre-existing transport machinery is activated, and a late phase (>2.5 h), which can be viewed as an anabolic action leading to a further amplification/differentiation of the Na+ transport machinery. At the transcriptional level, both early and late responses are initiated during the lag period, but the functional impact of newly synthesized regulatory proteins is faster than that of the structural ones. K-Ras2 and SGK were identified as the first early aldosterone-induced regulatory proteins in A6 epithelia. Their mRNAs also were shown to be regulated in vivo by aldosterone, and their expression (constitutively active K-Ras2 and wild-type SGK) was shown to increase the function of ENaC coexpressed in Xenopus oocytes. Recently, aldosterone was also shown to act on transcription factors in A6 epithelia: It down-regulates the mRNAs of the proliferation-promoting c-Myc, c-Jun, and c-Fos by a post-transcriptional mechanism, whereas it up-regulates that of Fra-2 (c-Fos antagonist) at the transcriptional level. Together, these new data illustrate the complexity of the regulatory network controlled by aldosterone and support the view that its early action is mediated by the induction of key regulatory proteins such as K-Ras2 and SGK. These early induced proteins are sites of convergence for different regulatory inputs, and thus, their aldosterone-regulated expression level tunes the impact of other regulatory cascades on sodium transport. This suggests mechanisms for the escape from aldosterone action.

In situ visualization of intratumor growth factor signaling: immunohistochemical localization of activated ERK/MAP kinase in glial neoplasms.

Abnormal growth factor signaling is implicated in the pathogenesis of gliomas. The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is a likely target, linking receptor tyrosine kinase activation to downstream serine/threonine phosphorylation events regulating proliferation and differentiation. Signaling within heterogeneous cell populations of gliomas cannot be adequately assessed by traditional biochemical enzyme assays. Immunohistochemical detection of doubly phosphorylated (activated) ERK/MAPK permitted visualization of spatially discrete cellular patterns of ERK/MAPK activation, compared with the relatively uniform expression of total ERK/MAPK protein. The astrocytic tumors, regardless of grade, had the highest overall degree of enzyme activation, whereas oligodendrogliomas had the least. Anaplastic progression in oligodendrogliomas resulted in a larger number of cells with active ERK/MAPK. Within glioblastomas, microvascular hyperplasia and necrosis were associated with ERK/MAPK activation in adjacent tumor cells. In addition to spatial patterns of intratumor paracrine signaling, a possible cell-cycle-associated regulation was detected: mitotic and actively cycling tumor cells showed diminished activation relative to cells in G0. Although ERK/MAPK activation was not restricted to neoplastic glia, consistent patterns of selective activation in tumor cells suggests that sustained activation may contribute to the neoplastic glial phenotype.

[Importance of oximetry parameters in the detection of hypoxia in the early phase of septic shock]. / Znacaj odnosa oksimetrijskih parametara u otkrivanju hipoksije u ranoj fazi septicnog soka.

The routinely monitored variables in shock stages include: arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. With vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. The therapeutic goal in septic shock stages is to maximize the values of cardiac index, O2 delivery (DO2) and O2 consumption (CO2). The aim of this study is to determine the relationship between O2 delivery and O2 consumption as an early sign of hypoxia. Fifteen patients with septic shock were treated in order to maximize the value of CI, DO2 and VO2. We compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after 24 hours. High levels of DO2 and VO2 do not exclude tissue hypoxia in the early stage of septic shock.