Class IVA Research Articles

RESEARCH ON CARDIAC ARRHYTHMIA AND HEART RATE VARIABILITY BASED ON 24- HOUR HOLTER ECG IN ELDERLY PATIENTS WITH DILATED CARDIOMYOPATHY

Objectives: Study several parameters of cardiac arrhythmia and heart rate variability in elderly patients with dilated cardiomyopathy (DCM), investigate the relationship and correlation between these parameters with NYHA functional heart failure class, EF, LVDd in elderly patients with DCM. Subjects and methods: 30 patients with DCM undergone treatment in Cardiovascular Department. Their ages were ≥ 60. These patients were diagnosed heart failure according to Framingham criteria and DCM according to WHO/ISFC criteria. 24-hour Holter ECG was the method used to analyse cardiac arrhythmia as well as heart rate variability (HRV). These parameters were compared with those considered normal of 81 people. Results: Sinus bradycardia accounted for 6.7%, sinus tachycardia 96.7%, sinus pause 13.3%, premature ventricular contraction 76.7%, supraventricular tachycardia 6.7%, ventricular tachycardia 40%. Lown’s classification: Lown’s class I undertook 46.7%, Lown’s class II 53.3%, Lown’s class III 23.3%, Lown’s class IVA 63.3%, Lown’s class IVB 36.7%, V 6.7%, p < 0.01. HRV indices: SDNN: 57.93 ± 16.99 ms, SDANN: 47.33 ± 16.69 ms, SDNNidx: 31.83 ± 11.00 ms, rMSSD: 19.87 ± 4.39 ms, pNN50: 3.67 ± 2.88%, the parameters acquired from case group were lower than those of control group with statistical difference (p < 0.0001). HRV decreased in proportion to NYHA functional heart failure class and LVEF. There was proportional correlation between LVEF with SDNN (r=0.77, p < 0.01), SDANN (r=0.76, p<0.01), SDNNidx (r=0.66, p<0.01), rMSSD (r=0.58, p<0.01), and pNN50 (r=0.69, p<0.01). Conclusion: 24-hour Holter ECG was valuable in detecting cardiac arrhythmia and HRV in elderly patients with DCM. This method also contributed to the following up, treatment and prognosis of this disease. Keywords: Arrhythmia, heart rate variability, dilated cardiomyopathy, elderly

Delayed complications after Gamma Knife surgery for intractable epilepsy

Despite the controversy concerning the clinical usefulness of Gamma Knife surgery (GKS; Elekta AB, Stockholm, Sweden) for intractable epilepsy, this treatment modality has attracted attention due to its low invasiveness. We report the long-term outcomes of four patients, focusing particularly on the efficacy and complications of GKS. We reviewed the data of four patients with medically intractable epilepsy who underwent GKS between 1998 and 2000 at our hospital. The marginal dose to the 50% isodose line was 24Gy in one patient and 20Gy in the remaining three patients. Two of the four patients were treated in the right temporal lobe, one was treated in the left parietal lobe, and one was treated in the right frontal lobe. The mean follow-up was 12.5years (range 12–14years). One patient was seizure free (Engel class IA) 24months after GKS, and two patients failed to show any seizure reduction (Engel class IVA). However, a clear aggravation was evident in one patient (Engel class IVC). All four patients underwent resective surgery due to radiation necrosis (RN) 7, 10, 10 and 12years after GKS. Three patients were seizure free (Engel class IA), and one was considered to have Engel class IB status following the resective surgery. GKS treatment resulted in insufficient seizure control and carried a significant risk of RN after several years. Drawbacks such as a delay in seizure control and the risk of RN should be considered when the clinical application of this treatment is evaluated.

Efficacy and safety of a new robot-assisted stereotactic system for radiofrequency thermocoagulation in patients with temporal lobe epilepsy

The aim of the present study was to evaluate the efficacy and safety of a newly developed robot-assisted frameless stereotactic system for deep electrode implantation and radiofrequency thermocoagulation (RFTC). Deep-electrode implantation was performed in the bilateral mesial temporal lobes of seven patients. Following the implantation of the deep electrodes through the monitored designed path, the epileptogenic zones were determined with the assistance of a robot system. Deep electrode electroencephalograms were recorded prior to and following RFTC. Treatment outcomes were evaluated by computed tomography scans and Engel classification criteria. The procedure was well tolerated by all patients with no patients suffered from severe permanent complications. After follow-ups for 34–62 months, four patients achieved Engel class I, including three patients with Ia classification, two patients were classified as Engel class IVa and one patient was classified as Engel class IVc. Therefore, robot-assisted frameless stereotaxy for deep electrode implantation and RFTC is indicated to be a safe and effective method that may be used effectively in clinical practice.

AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from −83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at −29/−17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

OP0064 Joint EULAR/ERA-EDTA recommendations for the management of adult and pediatric lupus nephritis

BackgroundThere is increasing evidence from clinical trials on which to base a rational approach to the care of lupus nephritis (LN).ObjectivesTo develop recommendations for the management of LN.MethodsQuestions were compiled...

Expression of phosphatase of regenerating liver family genes during embryogenesis: an evolutionary developmental analysis among Drosophila, amphioxus, and zebrafish

BackgroundPhosphatase of regenerating liver (PRL) family is classified as class IVa of protein tyrosine phosphatase (PTP4A) that removes phosphate groups from phosphorylated tyrosine residues on proteins. PRL phosphatases have been implicated in a number of tumorigenesis and metastasis processes and are highly conserved. However, the understanding of PRL expression profiles during embryonic development is very limited.ResultsIn this study, we demonstrated and characterized the comprehensive expression pattern of Drosophila PRL, amphioxus PRL, and zebrafish PRLs during embryonic development by either whole mount immunostaining or in situ hybridization. Our results indicate that Drosophila PRL is mainly enriched in developing mid-guts and central nervous system (CNS) in embryogenesis. In amphioxus, initially PRL gene is expressed ubiquitously during early embryogenesis, but its expression become restricted to the anterior neural tube in the cerebral vesicle. In zebrafish, PRL-1 and PRL-2 share similar expression patterns, most of which are neuronal lineages. In contrast, the expression of zebrafish PRL-3 is more specific and preferential in muscle.ConclusionsThis study, for the first time, elucidated the embryonic expression pattern of Drosophila, amphioxus, and zebrafish PRL genes. The shared PRL expression pattern in the developing CNS among diverse animals suggests that PRL may play conserved roles in these animals for CNS development.

The case of 17-year-old male with LEOPARD syndrome

LEOPARD syndrome is a phenotypic expression of mutations in several genes: PTPN11, RAF1, and BRAF. All these genes are responsible for Ras/MARK signaling pathway, which are important for cell cycle regulation, differentiation, growth, and aging. Mutations result in anomalies of skin, skeletal, and cardiovascular systems. The LEOPARD syndrome means lentigines, electrocardiographic conducting abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retarded growth, and deafness. Mutations affect tyrosine proteases, which are included in the signal pathway between the cell membrane and the nucleus. This rare autosomal dominant disorder is characterized by high variability of clinical manifestations. Usually only lentigines are common. Clinical diagnosis is based on lentigines and 2 other symptoms; in cases without lentigines – 3 symptoms and at least one affected first-line relative. Herein, we report the case of 17-year-old male who had idiopathic hypertrophic cardiomyopathy with left ventricular obstruction, and supraventricular and ventricular extasystoles, class IVa, left bundle branch block, as a life-threatening manifestation of LEOPARD syndrome. For the treatment of cardiac manifestations of this syndrome, the patient underwent two interventions: (1) mitral valve replacement by mechanical valve Optiform number 27 with surgical resection of left ventricular outflow tract and subaortic membrane excision; (2) implantable cardioverter-defibrillator therapy.<Learning objective: Explain the abbreviation L.E.O.P.A.R.D. (Lentigines, Electrocardiographic conducting abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retarded growth, and Deafness). Suspect the signs of L.E.O.P.A.R.D.-syndrome. Realize the etiology. Evaluate probability of this congenital disease on the ground of the clinical manifestations and laboratory data. Measure the significance for health of changes of organs and systems. Choose the main and dangerous manifestation of L.E.O.P.A.R.D.-syndrome. Select the best way for treatment such patients.>

Identification of a Coding Sequence and Structure Modeling of a Glycine-Rich RNA-Binding Protein (CmGRP1) from Chelidonium majus L.

The family of glycine-rich plant proteins (GRPs) is a large and complex group of proteins that share, as a common feature, the presence of glycine-rich domains arranged in (Gly)n-X repeats that are suggested to be involved in protein–protein interactions, RNA binding, and nucleolar targeting. These proteins are implicated in several independent physiological processes. Some are components of cell walls of many higher plants, while others are involved in molecular responses to environmental stress, and mediated by post-transcriptional regulatory mechanisms. The goals of this study are to identify the coding sequence of a novel glycine-rich RNA-binding protein from Chelidonium majus and to propose its structural model. DNA fragments obtained using degenerate PCR primers showed high sequence identities with glycine-rich RNA-binding protein coding sequences from different plant species. A 439-bp nucleotide sequence is identified coding for a novel polypeptide composed of 146 amino acids, designated as CmGRP1 (C. majus glycine-rich protein 1), with a calculated MW of 14,931 Da (NCBI GenBank accession no. HM173636). Using NCBI CDD and GeneSilico MetaServer, a single conserved domain, the RNA recognition motif (RRM), was detected in CmGRP1. The C-terminal region of CmGRP1 is a glycine-rich motif (GGGGxxGxGGGxxG), and it is predicted to be disordered. Based on a 1fxl crystal structure, a 3D model of CmGRP1 is proposed. CmGRP1 can be classified as a class IVa plant GRP, implicated to play a role in plant defense.

Effect of the GWDA compost and PRP sol fertilisation on the total content of nickel, manganese and lead and their soluble forms in soil

A field experiment was carried out at the Agricultural Experimental Station in Lipnik near Stargard Szczecinski in 2008-2009. It was set up on soil classified as soil quality class IVa and good rye complex (5). In the study, the GWDA compost produced at the Municipal Sewage Treatment Plant in Stargard Szczecinski was used. This compost was characterised by neutral reaction (pH H2O 7.15). The total content of heavy metals, which limits its possible use as fertiliser, did not exceed the standards given in the Regulation of the Minister of Agriculture and Rural Development (Official Journal of Law No. 119, item 765 of 2008). Compost doses were determined based on the total nitrogen content. It was assumed that 100 kg N ha –1 was added to soil with the 1 st dose, 200 kg N with the 2 nd dose, and 300 kg N with the 3rd dose. The experiment was carried out in two series: with and without addition of the active substance PRP SOL. In autumn 2007, respective compost doses were introduced into soil on established plots according to the experimental design. The active substance PRP SOL was applied at a 150 kg ha –1 dose before sowing or seeding test plants. The whole experimental area was fertilised with multicomponent fertiliser Polifoska 6 in a 200 kg ha–1 dose in 2008 and 2009. Due to a small content of nitrogen in Polifoska 6 (6% N), top-dressing nitrogen fertilisation was applied in the form of urea (46% N) at a 100 kg N ha –1 dose. The test plants were winter wheat cultivar Korweta in 2008 and spring rape cultivar Bosman in 2009. The results indicate that the total average manganese, lead and nickel content in soil increased by 15.1%, 5.0% and 3.0%, respectively, under organic fertilisation with and without the active substance compared to the initial content. In the course of time, manganese, nickel and lead content decreased to a different extent. This resulted from these microelements being taken up by the cultivated crop or their leaching outside the rhizo

FRAMELESS STEREOTACTIC PLACEMENT OF FORAMEN OVALE ELECTRODES IN PATIENTS WITH DRUG-REFRACTORY TEMPORAL LOBE EPILEPSY

Semi-invasive foramen ovale electrodes (FOEs) are used as an alternative to invasive recording techniques in the presurgical evaluation of patients with temporal lobe epilepsy. To maximize patient safety and interventional success, frameless stereotactic FOE placement by use of a variation of an upper jaw fixation device with an external fiducial frame, in combination with an aiming device and standard navigation software, was evaluated by the Innsbruck Epilepsy Surgery Program. Patients were immobilized noninvasively with the Vogele-Bale-Hohner headholder (Medical Intelligence GmbH, Schwabmünchen, Germany) to plan computed tomography and surgery. Frameless stereotactic cannulation of the foramen and intracranial electrode placement were achieved with the help of an aiming device mounted to the base plate of the headholder. Ease of applicability, safety, and results obtained with foramen ovale recording were investigated. Twenty-six FOEs were placed in 13 patients under general anesthesia. The foramen ovale was successfully cannulated in all patients. One patient reported transient painful mastication after the procedure as a complication attributable to use of the Vogele-Bale-Hohner mouthpiece. In one patient, a persistent slight buccal hypesthesia was present 3 months after the procedure. To pass the foramen, slight adjustments in the needle position had to be made in 10 sides (38.4%). To place the intracranial electrodes, adjustments were necessary six times (23.7%). An entirely new path had to be planned once (3.8%). Seizure recording provided conclusive information in all patients (100%). Outcome in operated patients was Engel Class Ia in six patients, Class IId in one patient, Class IIb in one patient, and Class IVa in one patient (minimum follow-up, 6 mo). The Vogele-Bale-Hohner headholder combined with an external registration frame eliminates the need for invasive head clamp fixation. FOE placement can be planned "offline" and performed under general anesthesia later. This can be valuable in patients with distorted anatomy and/or small foramina or in patients not able to undergo the procedure under sedation. Results are satisfactory with regard to patient safety, patient comfort, predictability, and reproducibility. FOEs supported further treatment decisions in all patients.

Koinzidenz von rasch progredienter dilatativer Kardiomyopathie und primärem Hyperparathyreoidismus: Verlauf vor und nach Entfernung eines Nebenschilddrüsenadenoms

Severe cardiac arrhythmias (Lown class IVa), rapid loss of physical capacity and dyspnoea on the slightest exertion occurred in a 55-year-old man with idiopathic dilated cardiomyopathy. In the preceding year he had recurrent diarrhoea and lost 23 kg in weight. He was found to have hypercalcaemia (3-3.2 mmol/l). The heart failure significantly improved under treatment with twice daily 12.5 mg captopril, 100 mg spironolactone daily, furosemide 40 mg twice daily, and digitoxin 0.07 mg daily. The arrhythmia responded to verapamil 80 mg and quinidine 160 mg, both drugs three times daily. Primary hyperparathyroidism was found to be the cause of the hypercalcaemia (parathormone 84 pmol/l). After the parathyroid adenoma had been removed the patient's condition again improved markedly. There were only rare monotopic extrasystoles, cardiac size regressed, and diuretics were no longer necessary. His medication at present is verapamil (80 mg three times daily), captopril (12.5 mg three times daily) and digitoxin (0.07 mg daily). It is concluded that the hypercalcaemia influenced the severity of the cardiomyopathy. It would seem that both intra- and extracellular calcium homoeostasis is of great importance in dilated cardiomyopathy.

Outcome of adult patients with temporal lobe tumours and medically refractory focal epilepsy

Tumours are a well-recognized cause of medically intractable epilepsies. Tumours represent the primary pathology in 10%-30% of patients undergoing surgical treatment for chronic epilepsy. This study examines the surgical and epileptological outcome of adults with temporal lobe tumoural epilepsy treated within a comprehensive epilepsy surgery programme. Between 1999 and 2004, 99 consecutive patients have been operated for intractable temporal lobe epilepsy (TLE). Among these, 14 adult patients exhibited temporal lobe neoplasms associated with TLE. Every patient underwent a comprehensive presurgical evaluation including video-EEG monitoring, MRI, interictal PET scan, ictal SPECT and neuropsychological testing. Surgical strategies were determined in an interdisciplinary seizure conference and tailored to the findings of the presurgical evaluation. All patients were available for follow up at regular intervals after 3, 6, 12 months and yearly thereafter. Epileptological outcome was classified according to Engel [10] and the ILAE (International League Against Epilepsy)/systems [33]. The surgical procedures performed were temporal lobe resection in 3 patients, extended lesionectomy in 4 and extended lesionectomy with resection of the temporomesial structures in 7. One patient with an astrocytoma grade III underwent a second and third operation for recurrent disease. Histological results: Astrocytoma 5 patients, ganglioglioma/gangliocytoma 5, oligodendroglioma 2, ependymoma 1 and dysembryoplastic neuroepithelial tumour (DNET) 1. Postoperative follow-up was performed after 12-74 months (mean 31). The outcome according to the Engel classification indicated class IA in 9 patients, class IC in 3, and 1 each in classes IIIA and IVA. Epileptological outcome according to the ILAE classification indicated class 1 (12 patients) and class 4 (2 patients). Surgical mortality was zero and mild permanent neurological deficits due to surgery were seen in 2 patients. Postoperatively 3 patients showed a homonymous quadrantanopia. Patients with drug resistant epilepsy and temporal lobe tumours should undergo evaluation in dedicated epilepsy surgery programmes.

Heightened Sensitivity to Paclitaxel in Class IVa β-Tubulin-transfected Cells Is Lost as Expression Increases

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of beta4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta4a-Tubulin differs from the more ubiquitous beta4b isotype at relatively few amino acid residues, yet beta4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta4a expression, beta4a and beta4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.

Comparison of β‐tubulin mRNA and protein levels in 12 human cancer cell lines

Antimitotic drugs are chemotherapeutic agents that bind tubulin and microtubules. Resistance to these drugs is a major clinical problem. One hypothesis is that the cellular composition of tubulin isotypes may predict the sensitivity of a tumor to antimitotics. Reliable and sensitive methods for measuring tubulin isotype levels in cells and tissues are needed to address this hypothesis. Quantitative measurements of tubulin isotypes have frequently relied upon inferring protein amounts from mRNA levels. To determine whether this approach is justified, protein and mRNA levels of beta-tubulin isotypes from 12 human cancer cell lines were measured. This work focused on only beta-tubulin isotypes because we had readily available monoclonal antibodies for quantitative immunoblots. The percentage of beta-tubulin isotype classes I, II, III, and IVa + IVb mRNA and protein were compared. For beta-tubulin class I that comprises >50% of the beta-tubulin protein in 10 of the 12 cell lines, there was good agreement between mRNA and protein percentages. Agreement between mRNA and protein was also found for beta-tubulin class III. For beta-tubulin classes IVa + IVb, we observed higher protein levels compared to mRNA levels.Beta-tubulin class II protein was found in only four cell lines and in very low abundance. We conclude that quantitative Western blotting is a reliable method for measuring tubulin isotype levels in human cancer cell lines. Inferring protein amounts from mRNA levels should be done with caution, since the correspondence is not one-to-one for all tubulin isotypes.

Alterations of β‐tubulin isotypes in breast cancer cells resistant to docetaxel

Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the beta-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in beta-tubulin isotypes in vitro and has sequenced the beta-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231, (oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of beta-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the beta-tubulin gene. At the mRNA level, class I, II, III, and IVa beta-tubulin mRNA isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI beta-tubulin mRNA isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa beta-tubulin mRNA. However, there were increased expressions in class II, IVb, and VI beta-tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in beta-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III beta-tubulin. This study demonstrates that altered expression of mRNA beta-tubulin isotypes and modulation of beta-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.

Treatment of diffuse proliferative lupus glomerulonephritis: A comparison of two cyclophosphamide-containing regimens

Cyclophosphamide (CYC) has proven beneficial in preserving renal function in patients with lupus with diffuse proliferative glomerulonephritis (DPGN). However, the optimal route of CYC administration is unknown because direct comparative studies are unavailable. In this open study, we compared the renal outcome of two historical cohorts of patients with diffuse proliferative lupus nephritis (World Health Organization classes IVa and IVb) treated with either intravenous (IV) pulse CYC (group A; n [equals] 22) or sequential oral CYC followed by azathioprine (AZA; group B; n [equals] 21) and followed up prospectively. Both groups of patients had similar clinical, biochemical, and renal parameters at baseline. At 24 months posttreatment, significant improvements in proteinuria, creatinine clearance, serum albumin level, and lupus serological results were evident in both groups. Compared with patients in group A, patients in group B had more complete or partial remission (90% versus 73%) and less risk for treatment failure (5% versus 14%), renal flares (5% versus 14%), and doubling of creatinine levels (5% versus 9%), but the difference was not statistically significant. However, patients treated with oral immunosuppression had an insignificant increase in rates of herpes zoster infection (19% versus 9%) and menstrual disturbance (50% versus 29%). We conclude that sequential oral immunosuppression with CYC and AZA tended to have better efficacy than IV pulse CYC in the treatment of lupus DPGN but was associated with more toxicities. Additional randomized trials involving a larger cohort of patients with a longer period of observation are necessary. [copy ] 2001 by the National Kidney Foundation, Inc.

Characterization of isotype-specific regions of five classes of canine beta-tubulin and their expression in several tissues and cell culture.

The structure of isotype-specific regions of classes 1, II, III, IVa and IVb of canine beta-tubulin was characterized by 3'-RACE and the expression of these isotypes in canine tissues was examined by ribonuclease protection assay (RPA). Furthermore, a malignant mammary tumor-derived osteosarcoma-like cell line was established and the altered expression of beta-tubulin isotypes in taxol-resistant sublines was analyzed. The deduced amino acid sequences in isotype-specific regions corresponding to classes I, II and IVb were identical to those of humans and mice, but those in classes III and IVa showed slight differences among species. RPA revealed that classes I and IVb were widely distributed, but classes II, III and IVa were restricted to the brain. Because RPA could clearly distinguish the expression of class IVa from that of class IVb, it was thought to be more useful than northern blot for analysis of beta-tubulin isotype expression. In vitro, taxol-resistant sublines displayed a significant increase in class IVa as compared with taxol-sensitive cells, suggesting that altered expression of class IVa was associated with taxol resistance in these cell lines.

Expression of Oncogenic Epidermal Growth Factor Receptor Family Kinases Induces Paclitaxel Resistance and Alters β-Tubulin Isotype Expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Social inequalities in health related behaviours in Barcelona

OBJECTIVEThis study describes social class inequalities in health related behaviours (tobacco and alcohol consumption, physical activity) among a sample of general population over 14 years old in Barcelona.DESIGNCross sectional study...

Differential expression of tubulin isotypes during the cell cycle.

Microtubules play an essential role in cell division. Little is known about possible variations of total tubulin and tubulin isotype expression during the cell cycle. We analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human MES-SA sarcoma cells. Although the total cellular tubulin content increases as the cells progress toward mitosis, the total tubulin/total protein ratio is stable during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of alpha, beta, and gamma-tubulin isotypes. Whereas alpha-tubulin isotype and gamma-tubulin transcripts were found to be expressed at constant levels throughout the cell cycle, some of the beta-tubulin isotype transcripts were found to be more highly expressed in dividing then in resting cells. Both of the class IV beta-tubulin isotype transcripts (human 5 beta and beta 2, Class IVa and IVb, respectively) were expressed in dividing K562 and MES-SA cells at twice the levels found in resting cells. Increased expression of the class IV isotype proteins in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA cells. A larger fraction of total cell tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total tubulin. These results show that total tubulin is expressed as constant levels throughout the cell cycle but that the degree of polymerization is increased as cells are committed to division. The relative overexpression of the two class IV beta-tubulin isotypes in dividing cells suggests functional specificity for these isotypes and a regulatory role of these isotypes on the microtubule network during mitosis.