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R Makuch

Publications and source records attributed to R Makuch.

At least 19 recordsLinked to original sources
PubMed2003

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus.

Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.

Actins↗
PubMed2001

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus.

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells. These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.

Acetylcholine↗
PubMed2000

Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study.

CONTEXT: Conventional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with a spectrum of toxic effects, notably gastrointestinal (GI) effects, because of inhibition of cyclooxygenase (COX)-1. Whether COX-2-specific inhibitors are associated with fewer clinical GI toxic effects is unknown. OBJECTIVE: To determine whether celecoxib, a COX-2-specific inhibitor, is associated with a lower incidence of significant upper GI toxic effects and other adverse effects compared with conventional NSAIDs. DESIGN: The Celecoxib Long-term Arthritis Safety Study (CLASS), a double-blind, randomized controlled trial conducted from September 1998 to March 2000. SETTING: Three hundred eighty-six clinical sites in the United States and Canada. PARTICIPANTS: A total of 8059 patients (>/=18 years old) with osteoarthritis (OA) or rheumatoid arthritis (RA) were enrolled in the study, and 7968 received at least 1 dose of study drug. A total of 4573 patients (57%) received treatment for 6 months. INTERVENTIONS: Patients were randomly assigned to receive celecoxib, 400 mg twice per day (2 and 4 times the maximum RA and OA dosages, respectively; n = 3987); ibuprofen, 800 mg 3 times per day (n = 1985); or diclofenac, 75 mg twice per day (n = 1996). Aspirin use for cardiovascular prophylaxis (</=325 mg/d) was permitted. MAIN OUTCOME MEASURES: Incidence of prospectively defined symptomatic upper GI ulcers and ulcer complications (bleeding, perforation, and obstruction) and other adverse effects during the 6-month treatment period. RESULTS: For all patients, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 0.76% vs 1.45% (P =.09) and 2. 08% vs 3.54% (P =.02), respectively. For patients not taking aspirin, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 0.44% vs 1.27% (P =.04) and 1.40% vs 2.91% (P =.02). For patients taking aspirin, the annualized incidence rates of upper GI ulcer complications alone and combined with symptomatic ulcers for celecoxib vs NSAIDs were 2.01% vs 2.12% (P =.92) and 4.70% vs 6.00% (P =.49). Fewer celecoxib-treated patients than NSAID-treated patients experienced chronic GI blood loss, GI intolerance, hepatotoxicity, or renal toxicity. No difference was noted in the incidence of cardiovascular events between celecoxib and NSAIDs, irrespective of aspirin use. CONCLUSIONS: In this study, celecoxib, at dosages greater than those indicated clinically, was associated with a lower incidence of symptomatic ulcers and ulcer complications combined, as well as other clinically important toxic effects, compared with NSAIDs at standard dosages. The decrease in upper GI toxicity was strongest among patients not taking aspirin concomitantly. JAMA. 2000;284:1247-1255

Aged↗
PubMed2000

Reversible changes in size of cell nuclei isolated from Amoeba proteus: role of the cytoskeleton.

Micrurgically isolated interphasal nuclei of Amoeba proteus, which preserve F-actin cytoskeletal shells on their surface, shrink after perfusion with imidazole buffer without ATP, and expand to about 200% of their cross-sectional area upon addition of pyrophosphate. These changes in size may be reproduced several times with the same nucleus. The shrunken nuclei are insensitive to the osmotic effects of sugars and distilled water, whereas the expanded ones react only to the distilled water, showing further swelling. The shrinking-expansion cycles are partially inhibited by cytochalasins. They are attributed to the state of actomyosin complex in the perinuclear cytoskeleton, which is supposed to be in the rigor state in the imidazole buffer without ATP, and to dissociate in the presence of pyrophosphate. Inflow of external medium to the nuclei during dissociation of the myosin from the perinuclear F-actin may be due to colloidal osmosis depending on other macromolecular components of the karyoplasm.

Actins↗
PubMed1999

Using sector endoluminal ultrasound to identify the normal pancreas when axial computed tomography is falsely positive.

RATIONALE AND OBJECTIVES: Little has been reported on the ability of endoluminal ultrasound (EUS) to identify a normal pancreas after an abnormal axial computed tomogram (CT). Many clinicians still use axial technology, as opposed to helical or spiral CT, which differ in scanning speed. Spiral CT and EUS are considered equal in their ability to diagnose pancreatic tumors. Although this is not the case with axial CT, the complementary role by EUS has not been defined. This study reports on the ability of EUS to identify the "true-negative" pancreas deemed abnormal by axial CT. METHODS: Sixty-five consecutive patients suspected of having a small pancreatic lesion were studied by comparing axial CT and EUS examinations, using each patient as his or her own control. Identification of a normal pancreas was reviewed using surgery, biopsy, and long-term clinical follow-up as the standard of truth. RESULTS: Thirty-three patients were documented as having small pancreatic lesions; the remaining 32 were normal. The sensitivity and specificity, respectively, were 91% and 41% for axial CT and 88% and 88% for EUS. The positive and negative predictive values, respectively, were 61% and 82% for CT and 88% and 88% for EUS. The statistical differences between axial CT and EUS were significant. CONCLUSION: An axial CT positive for a small pancreatic mass requires confirmation with additional imaging before invasive management. The specificity of EUS--twice that of CT--is strong evidence that EUS can fulfill this role. Review of the literature supports the conclusion that EUS should be required in the workup of small pancreatic lesions identified at axial CT.

Chronic Disease↗
PubMed1998

The first caldesmon-like protein in higher plants.

Using anti-caldesmon polyclonal and monoclonal (raised against the N-terminal fragment of chicken gizzard caldesmon) antibodies, a plant caldesmon-like protein, 107 kDa as determined by SDS-gel electrophoresis, has been identified based on Western blotting of total extracts of Ornithogalum virens pollen tubes. Biochemical investigations showed common properties of this protein with animal caldesmon--it binds to actin and, in a Ca(2+)-dependent manner, to calmodulin. In contrast to animal caldesmon, this plant cell counterpart is relatively resistant to proteolysis by endogenous proteases and sensitive to heat treatment. Our results show the presence of a caldesmon-like protein in higher plants for the first time.

Actins↗
PubMed1998

Verapamil use in patients with cardiovascular disease: an overview of randomized trials.

BACKGROUND: Several reports have questioned the lack of safety data on calcium antagonists as a drug class. Because this drug class is heterogeneous, unique features of certain calcium antagonists may set them apart in terms of safety and efficacy. HYPOTHESIS: With in excess of 7,000 person-years of observation from randomized clinical trials, verapamil was selected to evaluate whether there was evidence of harm in patients with cardiovascular disease. METHODS: MEDLINE search of English-language articles, Science Citation Index, Current Contents, manual review of cited references, pharmaceutical files, and investigator correspondence was performed. Independent review of 66 articles identified 14 randomized, parallel-group studies for inclusion. Independent, duplicate assessments were made of patient outcomes and trial characteristics (including study design, treatment dosage and schedule, duration of treatment, inclusion criteria, and sample size). Standard meta-analytic techniques were employed for analysis and interpretation of results. RESULTS: Based on over 4,000 person-years of observation, patients with acute myocardial infarction (MI) treated with verapamil had a decreased risk of nonfatal reinfarction compared with placebo (relative risk 0.79; 2-sided 95% confidence interval 0.65.0.97; p = 0.024). Verapamil had no significant effect on overall mortality compared with placebo (relative risk ranged from 0.93; 2-sided 95% confidence interval 0.78, 1.10; p = 0.40 to 0.86; 2-sided 95% confidence interval 0.71, 1.04; p = 0.13) depending on rules used to include or exclude patients from the pooling process. For the combined outcome of death or reinfarction, verapamil use was associated with a decreased risk compared with placebo (relative risk 0.82; 2-sided 95% confidence interval 0.70, 0.97; p = 0.016). In patients with angina involving a wide spectrum of disease severity, data were limited to 2,900 person-years of observation, and verapamil use did not appear to be associated with an apparent effect on mortality or MI. Data available from randomized studies of verapamil in patients with hypertension were too limited to reach conclusions (50 person-years of observation, with no deaths or MIs reported). Subgroups of hypertensive patients in two of the largest post-MI studies and the largest angina study, involving over 600 patients, yielded little useful added information. CONCLUSIONS: In patients with MI, the risks of both nonfatal reinfarction and the combined outcome of death or nonfatal MI were reduced over intermediate-term follow-up among patients treated with verapamil compared with controls (p = 0.024 and p = 0.016, respectively). In patients with angina, no evidence for harm was noted, but in hypertension the data were too limited to draw conclusions. These findings support the need to distinguish among different calcium antagonist compounds and to emphasize the need for more data in patients with hypertension.

Angina Pectoris↗
PubMed1997

Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.

We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.

Animals↗
PubMed1997

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.

Actins↗
PubMed1996

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations.

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guinea-pig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

Animals↗
PubMed1996

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon.

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

Actins↗
PubMed1996

Variations in the natural history and survival of patients with supratentorial low-grade astrocytomas.

Data from 55 consecutive patients with low-grade astrocytomas treated between 1982 and 1990 were analyzed to determine specific outcome factors, including time to recurrence, incidence of anaplastic transformation, and survival. Gender, type of symptoms, contrast enhancement, and timing of radiation therapy were not significant in determining outcome. Patients who had symptoms for > 2 years and underwent gross-total resection of the tumor, with age as a continuous variable, were associated with significantly longer time to recurrence and survival. Within the population of patients with low-grade astrocytomas, patients with chronic epilepsy clearly had the best prognoses. There were no tumor recurrences or deaths in 27 patients with chronic epilepsy, regardless of the extent of surgery and without the use of radiotherapy. Ten-year survival was 100% for 31 patients who underwent gross-total tumor resection, regardless of the length of preoperative symptoms. Immediate postoperative radiotherapy did not prolong the time to recurrence, reduce the incidence of transition to more malignant tumors at recurrence, or increase the length of survival when compared with delayed radiotherapy. Because recurrence with a high-grade lesion caused 92% of the mortality in our series, the benefit in patients who underwent aggressive surgery seems to result from a significant decrease in the risk of recurrence when compared with patients who underwent anything less than gross-total resection. Our data also suggest that variability in the natural history of low-grade astrocytomas has a strong influence in determining survival and that tumors associated with chronic epilepsy are much less likely to become more malignant over time.

Adult↗
PubMed1996

Quantitative comparison of planar and SPECT normal data files of thallium-201, technetium-99m-sestamibi, technetium-99m-tetrofosmin and technetium-99m-furifosmin.

UNLABELLED: In recent years, several of 99mTc-labeled myocardial perfusion imaging agents have been developed, such as 99mTc-sestamibi, 99mTc-tetrofosmin and 99mTc-furifosmin. Although images obtained with these new tracers have a general similar appearance, there are differences in the myocardial kinetics, body distribution, general quality of images and imaging protocols. The aim of this study was to quantitatively compare normal exercise planar and SPECT data files obtained with 201TI and 99mTc-labeled agents. METHODS: Lower-limit-of-normal curves were generated for each specific radiopharmaceutical from normal subjects with low (< 3%) pretest likelihood of coronary artery disease using circumferential count distribution profiles from planar and SPECT exercise images. Lower-limit-of-normal curves were statistically compared using the nonparametric Kruskall-Wallis and Wilcoxon tests. RESULTS: Planar and SPECT lower-limit-of-normal curves generated for each radiopharmaceutical showed general similarities. Statistically significant differences among the lower-limit-of-normal curves were found in the planar left anterior oblique view and in the planar left lateral view (p < 0.05 for each). No statistically significant differences existed between lower-limit-of-normal curves of various radiopharmaceuticals on the planar anterior view and on SPECT imaging. CONCLUSION: For quantitative analysis of planar images, radiopharmaceutical-specific normal data files are mandatory. Although SPECT normal data files of various radiopharmaceuticals are not statistically different, they are not identical. It appears, nevertheless, prudent to use radiopharmaceutical-specific normal data files for quantitative analysis of SPECT images.

Adult↗
PubMed1995

Interaction of calponin with actin and its functional implications.

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Actins↗
PubMed1994

Polymerization of actin induced by actin-binding fragments of caldesmon.

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Actins↗
PubMed1994

Neoadjuvant chemotherapy for advanced ovarian cancer.

The purpose of this study was to compare the survival of women with clinical, radiologic, and histologic findings compatible with advanced ovarian cancer who were treated with neoadjuvant chemotherapy to that of a group of women with FIGO stage IIIC (suboptimal debulking) and stage IV epithelial ovarian cancers treated with the same chemotherapy. Eleven women with physical, radiologic, and histologic findings compatible with advanced ovarian cancer (median age, 73 years) treated with neoadjuvant carboplatin and cyclophosphamide chemotherapy were compared to 18 women (median age, 60 years) who had stage III and suboptimal surgical cytoreduction (> 2 cm residual tumor, 13 patients) or stage IV (5 patients) ovarian cancer followed by the same chemotherapy. The progression-free survival for the 11 women receiving neoadjuvant chemotherapy was 9.1 months which was not statistically different from the 8.5 months progression-free survival for the 18 women with suboptimally debulked stage IIIC or stage IV disease (P = 0.98). The overall survival was not significantly different (P = 0.26). Neoadjuvant-treated patients tended to tolerate chemotherapy better as none required dose reduction for bone marrow suppression, while 6 of 18 conventionally treated patient required dose reductions. In conclusion, neoadjuvant chemotherapy appears to be an effective means of palliating women with clinical, radiological, and histologic findings compatible with advanced ovarian cancer. A prospective study is necessary in which women with findings compatible with advanced ovarian cancer that is not likely to be effectively cytoreduced surgically are randomized to either undergo cytoreductive surgery followed by chemotherapy or receive neoadjuvant chemotherapy.

Adult↗