Arrhythmia and sudden death associated with elevated cardiac chloride channel activity.

The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents.

Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.

Raf-1 is a serine/threonine protein kinase that transduces signals from cell surface receptors to the nucleus. Interaction of Ras with a regulatory domain in the N-terminal half of Raf-1 is postulated to regulate Raf-1 protein kinase and signaling activities. To better understand molecular interactions of Ras with Raf-1 and regulation of the Raf-1 kinase, a panel of Raf-1 N-terminal mutants expressed in the baculovirus-insect cell system was used for mapping the precise region necessary for Ras interaction in the context of full-length, functional Raf-1 kinase. An 80-amino-acid sequence in Raf-1 between positions 53 and 132 was found to confer the ability to bind Ras protein in vitro and in infected insect cells. Deletion of residues 53 to 132 abolished Raf-1 kinase activation by Ras in insect cells, indicating that activation of the Raf-1 kinase by Ras requires the capacity to physically interact with Ras. By contrast, deletion of this Ras-binding site did not diminish activation of Raf-1 kinase by Src, implying that Src and Ras can activate Raf-1 through independent mechanisms. Significantly, Raf-1 mutants lacking the entire zinc finger motif or containing substitutions of two critical cysteine residues in the zinc finger retained the ability to bind Ras and to be activated by this interaction. Consistent with results obtained in the baculovirus-insect cell system, deletion of residues 53 to 132 but not mutations in the zinc finger motif abrogated the ability of kinase-inactive, dominant negative Raf-1 to block Ras-mediated signaling in Xenopus oocytes. Together, these results provide evidence that the direct physical interaction of Ras with Raf-1 amino acids 53 to 132 is required for activation of the Raf-1 kinase and signaling activities by Ras but not by Src. Furthermore, the adjacent zinc finger motif in Raf-1 is not essential either for interaction with Ras or for activation of the Raf-1 kinase.

Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for gene therapy.

It has been well demonstrated that interleukin-12 (IL-12) could be useful to defend against a variety of pathogens, to suppress tumor growth and metastasis, and even to be employed as an adjuvant of vaccines to enhance beneficial type 1 T helper (Th1) cell response over detrimental type 2 T helper (Th2) cell responses. To apply IL-12 genes in gene therapy such as a DNA vaccine, a pIL-12 vector was constructed that contained two cytomegalovirus (CMV) promoters to drive the expression of p35 and p40 subunits, respectively. In addition, a pscIL-12 vector was designed with a linker to fuse p35 cDNA with p40 cDNA to produce a single-chain IL-12 protein, ensuring not only that the expression of p35 and p40 subunits was equally expressed, but also that no free p40 subunits interfered with IL-12 activity. The data suggested pIL-12 could produce a rather high level of biologically active IL-12 after transfection of COS cell lines as well as C2C12 muscle cell lines, as measured by both concanavalin A blast proliferation assay and enzyme-linked immunosorbent assay. Interestingly, the pscIL-12 vector could also express a bioactive murine IL-12 fusion protein in vitro. Furthermore, in vivo functional studies also demonstrated that mice co-immunized with a pS vector expressing the major envelope protein of hepatitis B virus (HBV) and IL-12 vectors encoding native IL-12 or single-chain IL-12 fusion protein elicited higher levels of IgG2a anti-HBs antibody and of Th1-related cytokine. Because p35 and p40 genes can be expressed in a vector by using a single promoter, pscIL-12 should be useful in future applications for nucleic acid vaccination or for gene therapy against diseases.

Prostate-specific targeting using PSA promoter-based lentiviral vectors.

The prostate-specific antigen (PSA) promoter is known to be highly tissue specific. Although its tissue specificity has been confirmed, its efficiency of gene transcription is significantly lower compared to known nonspecific viral promoters. These lower levels of promoter activity therefore pose a problem when developing an efficacious gene vector for prostate cancer gene therapy. Thus, selecting an appropriate therapeutic gene and vector system to carry the gene driven by the PSA promoter (PSAP) is important. In the studies described here, a human immunodeficiency virus (HIV)-1-based lentiviral vector carrying either the enhanced green fluorescent protein (EGFP) reporter or the diphtheria toxin A (DTA) gene was constructed. The results demonstrate that the PSA promoter in a lentiviral vector drives genes in prostate cells with satisfactory efficacy and specificity. The tissue-specific expression of the DTA protein efficiently eradicates LNCaP prostate cells in culture. We also infected prostate cancer cells and control cells carried by nude mice with the EGFP lentiviral vector. Significant numbers of EGFP-positive LNCaP cells were detected in all the mice bearing these tumors, but no EGFP-positive control cells were detected in any other mouse tissue. The high levels of expression in prostate cells, compared with the low levels of background expression in other cells, show that the PSAP-lentiviral vector could be a potential useful tool for gene therapy of metastatic prostate cancer.

Regulation of glucocorticoid receptor function through assembly of a receptor-heat shock protein complex.

Incubation of immunopurified, hormone-free mouse glucocorticoid receptors with rabbit reticulocyte lysate results in ATP-dependent and monovalent cation-dependent assembly of the GR into a heterocomplex with hsp90, hsp70, and hsp56. Heterocomplex assembly is accompanied by conversion of the receptor from a form that does not bind steroid to a high affinity steroid-binding conformation. Reticulocyte lysate also promotes ATP-dependent dissociation of unliganded receptors from a prebound receptor-DNA complex. Receptor released from DNA has been reconstituted into the heat shock protein heterocomplex and converted to the non-DNA-binding state. The reticulocyte lysate also reconstitutes pp60v-src into a heterocomplex containing hsp90 and p50, both of which are components of the native heterocomplex form of the tyrosine kinase in cytoplasm. Although the c-Raf-1 serine/threonine kinase has never been found in native association with hsp90, it can be assembled into a heat shock protein heterocomplex by the ATP-dependent system in reticulocyte lysate.

Screening for hypercholesterolaemia in patients with other risk factors for coronary heart disease.

OBJECTIVE: To determine the prevalence of hypercholesterolaemia in individuals with other major risk factors of coronary heart disease and markers of hypercholesterolaemia. METHODS: A community-based cross-sectional study on a random sample of 261 persons aged between 35 and 69 years residing in the flatted housing estate of West Coast, Singapore, was conducted in 1997. A questionnaire, together with a medical examination and investigation involving the use of Reflotron machine, were used to collect the information required. RESULTS: 43.2% of the population had at least 1 major risk factor, with 18.1% being smokers, 18.1% with hypertension, 6.5% with diabetes mellitus, 5.1% being obese, and 5.0% with a family history of coronary heart disease. Higher percentages of individuals with hypercholesterolaemia were found when each risk factor was present. 9.5% had 2 or more risk factors, of which, 21.1% had high cholesterol levels. A high prevalence of hypercholesterolaemia that was statistically significant was found amongst subjects with corneal arcus below the age of 60. CONCLUSIONS: The risk factors of coronary heart disease remain prevalent in our population. We recommend screening for serum cholesterol only in those with at least 2 major risk factors of coronary heart disease in the general population between 35 and 69 years of age.

Development of an electronic emergency department-based geo-information injury surveillance system in Hong Kong.

OBJECTIVES: To describe the experience in the development of an electronic emergency department (ED)-based injury surveillance (IS) system in Hong Kong using data-mining and geo-spatial information technology (IT) for a Safe Community setup. METHODS: This paper described the phased development of an emergency department-based IS system based on World Health Organization (WHO) injury surveillance Guideline to support safety promotion and injury prevention in a Safe Community in Hong Kong starting 2002. RESULTS: The initial ED data-based only collected data on name, sex, age, address, eight general categories of injury types (traffic, domestic, common assault, indecent assault, batter, industrial, self-harm and sports) and disposal from ED. Phase 1--manual data collection on International Classification of External Causes of Injury pre-event data; Phase 2--manual form was converted to electronic format using web-based data mining technology with built in data quality monitoring mechanism; Phase 3--integration of injury surveillance-data with in-patient hospital information; and Phase 4--geo-spatial information and body mapping were introduced to geo-code exact place of injury in an electronic map and site of injury on body map. CONCLUSION: It was feasible to develop a geo-spatial IS system at busy ED to collect valuable information for safety promotion and injury prevention at Safe Community setting. The keys for successful development and implementation involves engagement of all stakeholders at design and implementation of the system with injury prevention as ultimate goal, detail workflow planning at front end, support from the management, building on exiting system and appropriate utilisation of modern technology.

Survey on medical records and EHR in Asia-Pacific region: languages, purposes, IDs and regulations.

OBJECTIVES: To clarify health record background information in the Asia-Pacific region, for planning and evaluation of medical information systems. METHODS: The survey was carried out in the summer of 2009. Of the 14 APAMI (Asia-Pacific Association for Medical Informatics) delegates 12 responded which were Australia, China, Hong Kong, India, Indonesia, Japan, Korea, New Zealand, the Philippines, Singapore, Thailand, and Taiwan. RESULTS: English is used for records and education in Australia, Hong Kong, India, New Zealand, the Philippines, Singapore and Taiwan. Most of the countries/regions are British Commonwealth. Nine out of 12 delegates responded that the second purpose of medical records was for the billing of medical services. Seven out of nine responders to this question answered that the second purpose of EHR (Electronic Health Records) was healthcare cost cutting. In Singapore, a versatile resident ID is used which can be applied to a variety of uses. Seven other regions have resident IDs which are used for a varying range of purposes. Regarding healthcare ID, resident ID is simply used as healthcare ID in Hong Kong, Singapore and Thailand. In most cases, disclosure of medical data with patient's name identified is allowed only for the purpose of disease control within a legal framework and for disclosure to the patient and referred doctors. Secondary use of medical information with the patient's identification anonymized is usually allowed in particular cases for specific purposes. CONCLUSION: This survey on the health record background information has yielded the above mentioned results. This information contributes to the planning and evaluation of medical information systems in the Asia-Pacific region.

The -844C/T polymorphism in the Fas ligand promoter associates with Taiwanese SLE.

FasL expression is critical in T-cell activation-induced apoptosis, which is involved in lupus pathogenesis. This study identified two SNPs in the FasL promoter regions from -1145 to -45 by genomic DNA sequencing. The -844C/T polymorphism was previously described by its location in and affect on the CCAAT/enhancer-binding protein beta (C/EBPB beta)-binding site and the other (-1094A/C, a novel polymorphism) was located at the NF-kappaB transcription-binding site. FasL gene promoter polymorphisms were genotyped in 260 systemic lupus erythematosus (SLE) patients and 280 healthy controls using MassArray matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The distribution of FasL promoter -844C/C genotype, predominant in Taiwanese, was skewed in Taiwanese SLE patients (odds ratio: 1.53; P-value=0.014). FasL promoter -844C/T polymorphism genotype distributions of Taiwanese, African Americans, and Caucasians differed. Moreover, no particular clinical association of -844C/T and -1094A/C polymorphisms with SLE was found in patients in Taiwan. This study confirmed that -844C/C genotype is associated with lupus susceptibility. The -1094A/C polymorphism is not significantly associated with lupus disease susceptibility, albeit the role of NF-kappaB pathway in FasL promoter activation remains unclear. Fas/FasL pathway may contribute to SLE polygenic disease entity.

Previous cesarean section and abortion as risk factors for developing placenta previa.

OBJECTIVE: To determine the risk of subsequent occurrence of placenta previa in women with a history of previous cesarean sections and/or spontaneous and induced abortions. METHODS: A retrospective analysis of all single gestation deliveries at National University Hospital of Singapore from 1993-1997 was done. Women with placenta previa were identified by clinical or ultrasonographic diagnosis. RESULTS: Of the 16,169 singleton deliveries, 164 women (1.0%) had placenta previa. Women with placenta previa had a significantly higher incidence of previous cesarean sections (p < 0.001). Among the 164 women with placenta previa, women with 1, 2, and 3 previous cesarean sections had 2.2 (95% CI 1.4, 3.4), 4.1 (95% CI 1.9, 8.8) and 22.4 (95% CI 6.4, 78.3) times increased risk of developing placenta previa respectively. Similarly, women with 2 or more previous abortions had a 2.1 (95% CI 1.2, 3.5) times increased risk of subsequently developing placenta previa. CONCLUSION: There is a strong association between previous cesarean section and risk of subsequent development of placenta previa. This risk increased with the number of previous cesarean sections. Increasing frequency of abortions was also found to predispose a woman to placenta previa.

Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel alpha subunit gene.

To investigate the regulation of the amiloride-sensitive epithelial sodium channel (ENaC) expression, we have characterized the genomic structure and performed promoter analyses of the alpha subunit of the human (h) ENaC gene. Genomic clones containing the alphahENaC gene were isolated and subjected to restriction-mapping analysis. The alphahENaC gene was shown to be composed of 13 exons and 12 introns. Primer extension analysis confirmed that transcription initiation occurred at the beginning of the reported alphahENaC cDNA, but also indicated potential heterogenous initiation sites. Examination of a 3.1 kb 5' flanking sequence revealed a notable absence of CCAAT or TATA-like elements but suggested three GC boxes and several putative transcription factor binding sites, including a glucocorticoid response element (GRE) consensus. A 250 bp minimal promoter was capable of directing expression of a secreted alkaline phosphatase reporter. This promoter activity was enhanced 2.5- and 4-fold by upstream flanking sequences. Dexamethasone treatment induced levels of expression from the longer, GRE-containing promoter fragments from 8- to 20-fold, but not from the minimal promoter. Precise deletion of the 15-bp, dyad GRE sequence completely abolishes the response of reporter expression to dexamethasone induction. These experiments indicate that glucocorticoid augmentation of lung epithelial Na+ transport occurs, at least in part, by direct stimulation of transcription of the ENaC genes.

Regulated expression of the human CFTR gene in epithelial cells.

We developed an epithelium-specific, inducible cystic fibrosis transmembrane conductance regulator (CFTR) expression system. In this system we used a human cytokeratin 18 expression cassette to drive epithelium-specific expression of the reverse tetracycline transactivator (rtTA), which turns on CFTR expression from a Tet-inducible promoter in the presence of doxycycline. CFTR expression was monitored by reverse-transcription polymerase chain reaction, immunostaining, and Western blotting. We confirmed that protein expression was dose-dependent in double stable transfected cell lines, with no detectable protein in the absence of doxycycline. However, low levels of CFTR mRNA could be detected in the uninduced state. When clones capable of inducing high levels of CFTR expression were analyzed, we observed a decrease in cell proliferation, consistent with reports in other cell lines (NIH3T3 and BTS). We generated transgenic mice expressing rtTA from the K18 expression cassette and demonstrated that the system retained its tissue specificity for lacZ reporter expression in vivo. When mice were induced with doxycycline, high levels of expression were found in the trachea, upper bronchi, and submucosal glands. Therefore, this inducible system can improve our understanding of the role of CFTR in the lung and should help in the design of safe and effective CF therapies.

The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase.

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras.Raf, Ras.Src, Raf.Mek, and Raf.Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf.hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.

Targeting transgene expression to airway epithelia and submucosal glands, prominent sites of human CFTR expression.

Targeting therapeutic gene expression to disease-affected tissues is an essential component of effective and safe gene therapy. After birth, CFTR gene expression in human lungs is localized predominantly in the epithelial cells lining the upper airways, especially in the ducts and serous tubules of the submucosal glands. We have developed a K18 expression cassette, based on the DNA control elements of the human cytokeratin 18 gene. Temporal and spatial analyses of transgenic mice demonstrated that this expression cassette targets transgene expression to almost all cell types in which CFTR is expressed. Airway epithelium expression started as early as 11.5 days of gestational age and continued into the adulthood of the transgenic mice. In these adult mice, the pattern of the reporter expression strikingly matched that of the human cytokeratin 18 and human CFTR genes. The transgene expression was epithelium-specific and undetectable in connective tissue, muscle, bone, cartilage, blood, and endothelial cells. Significantly, high levels of expression were detected in tracheal submucosal glands. Together, these results suggest that our K18 expression cassette has a high potential for clinical application in gene therapy for patients with cystic fibrosis.

Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.

Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

Apparent extension of the atrioventricular interval due to sensor-based algorithm against supraventricular tachyarrhythmias.

Rapid ventricular tracking response to supraventricular tachyarrhythmia is one major limitation to DDD pacing. In a DDDR pacemaker, sensor-based algorithms have been used to control these arrhythmias. These include the use of an interim rate limit (conditional ventricular tracking limit) or a separate maximum tracking and sensor rate limits (discrepant upper rate). These algorithms limit inappropriate ventricular pacing rate during tracking of pathological supraventricular tachyarrhythmia and atrial flutter by Wenckebach-like prolongation of the AV interval. We observed that this may cause an unexpected extension of the AV interval in patients with high atrial rate and intact AV nodal conduction. This was due to P wave rate above the conditional ventricular tracking limit or maximum tracking limit, but AV paced interval prolongation was avoided by the occurrence of intrinsic conduction, albeit at an AV interval longer than the programmed AV interval. This might appear as failure of ventricular pacing on the ECG. This phenomenon is a modified form of "upper rate" behavior occurring in the AV interval, and should be recognized as a normal behavior rather than pacemaker malfunction.

Quantitative comparison of rate response and oxygen uptake kinetics between different sensor modes in multisensor rate adaptive pacing.

Although multisensor pacing may mitigate the inadequacy of rate adaptation in a single sensor system, the clinical role of multisensor driven rate adaptive pacing remains unclear. The cardiopulmonary performance of six patients (mean age 63.5 +/- 10 years) who had undergone the implant of combined QT and activity VVIR (Topaz) pacemakers was assessed during submaximal and maximal treadmill exercise with the rate response sensor randomly programmed to either single sensor mode, QT and activity (ACT), or dual sensor mode, with equal contribution of QT and ACT (QT = ACT). The rate of response, the proportionality, oxygen kinetics, and maximal exercise performance of the various sensor modes during exercise were measured and compared. The ACT sensor mode "overpaced" and the QT and QT = ACT sensor modes "underpaced" during the first three quartiles of exercise (P < 0.05). The ACT sensor mode also gave the fastest rate of response with the shortest delay (13 +/- 1.5 sec vs 145 +/- 58 sec and 41 +/- 17 sec, P < 0.05), time to 50% rate response (39 +/- 2.7 sec vs 275 +/- 48 sec and 203 +/- 40 sec, P < 0.05), and time to 90% of rate response (107 +/- 21 sec vs 375 +/- 34 sec and 347 +/- 34 sec, P < 0.05) and a smaller oxygen debt (0.87 +/- 0.16 L vs 1.10 +/- 0.2 L and 1.07 +/- 0.18 L, P < 0.05) compared to the QT and QT = ACT sensor modes, respectively. These differences were most significant at low exercise workloads. Thus, different sensor combinations result in different rate response profiles and oxygen delivery, especially during low level exercise.(ABSTRACT TRUNCATED AT 250 WORDS)