Biomedical subjects

A Heller

Publications and source records attributed to A Heller.

At least 55 records · Page 3Linked to original sources

PubMed2000

Evidence of elevated intracellular calcium levels in weaver homozygote mice.

1. A mutation in the G-protein-linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2. We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild-type neurons. 3. High-K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild-type neurons. 4. weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX-314. 5. QX-314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6. These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

Animals↗

PubMed2000

Expression of a PKR dominant-negative mutant in myogenic cells interferes with the myogenic process.

In this study, we explored the possibility that PKR, a dsRNA-activated regulatory protein, is an essential component in the differentiation program of myogenic cells in vitro. For this purpose, we used a retroviral expression vector pMV7, harboring the PKR dominant-negative mutant PKRDelta6 (pMV7-p68Delta6). Murine C2C12 myogenic cells were transfected either with pMV7 or with pMV7-p68Delta6. Neomycin-resistant clones from both types were isolated and expanded and the results obtained with the representative clones C2-NEO (transfected with pMV7) and clone 17 and clone 22 (both transfected with pMV7-p68Delta6) are presented. In clone 17 and 22 cells, regardless of IFN treatment, a similar level of the transfected human p68 PKR mutant was detected. This protein was absent in C2-NEO cells. In parallel, in all types of cells, a low basal level of the endogenous murine p65 PKR protein was observed, which was further induced by IFN. However, PKR enzymatic activity was significantly induced by IFN only in C2-NEO cells, while it was hardly detected in both clones 17 and 22, even after IFN treatment. Furthermore, in contrast to C2-NEO cells, only a slight to moderate increase in enzymatic activity was observed in clone 17 and 22 differentiating cells. Next, cells were grown either in growth medium (GM) or differentiation medium (DM), and the progression of the myogenic program was studied. An inhibition in myotube formation in clone 17 versus C2-NEO cells cultivated in DM was clearly observed. Furthermore, while the growth rate and thymidine incorporation were reduced in C2-NEO cells grown in DM, both clone 17 and 22 cells were less affected under the same conditions. Similarly, a delay in the accumulation of the transcription factors MyoD and myogenin, as well as in creatine kinase activity and accumulation of troponin T, was detected in DM-cultivated clone 17 and clone 22 cells. Moreover, a delay in the induction of p21 (WAF1), in down-regulation of cyclin D1 and c-myc, and in the accumulation of the underphosphorylated form of pRb was also observed in clone 17 cells. We conclude that inhibition of endogenous PKR activity by a PKR dominant-negative mutant interferes with the myogenic program of murine C2C12 myogenic cells.

Animals↗

PubMed2000

Concordance of the toxicity of pharmaceuticals in humans and in animals.

This report summarizes the results of a multinational pharmaceutical company survey and the outcome of an International Life Sciences Institute (ILSI) Workshop (April 1999), which served to better understand concordance of the toxicity of pharmaceuticals observed in humans with that observed in experimental animals. The Workshop included representatives from academia, the multinational pharmaceutical industry, and international regulatory scientists. The main aim of this project was to examine the strengths and weaknesses of animal studies to predict human toxicity (HT). The database was developed from a survey which covered only those compounds where HTs were identified during clinical development of new pharmaceuticals, determining whether animal toxicity studies identified concordant target organ toxicities in humans. Data collected included codified compounds, therapeutic category, the HT organ system affected, and the species and duration of studies in which the corresponding HT was either first identified or not observed. This survey includes input from 12 pharmaceutical companies with data compiled from 150 compounds with 221 HT events reported. Multiple HTs were reported in 47 cases. The results showed the true positive HT concordance rate of 71% for rodent and nonrodent species, with nonrodents alone being predictive for 63% of HTs and rodents alone for 43%. The highest incidence of overall concordance was seen in hematological, gastrointestinal, and cardiovascular HTs, and the least was seen in cutaneous HT. Where animal models, in one or more species, identified concordant HT, 94% were first observed in studies of 1 month or less in duration. These survey results support the value of in vivo toxicology studies to predict for many significant HTs associated with pharmaceuticals and have helped to identify HT categories that may benefit from improved methods.

Animals↗

PubMed2000

Spiers Memorial Lecture. On the hypothesis of cathodic protection of genes.

Explore the source record for details and available documents.

DNA↗

PubMed2000

[The complement system: an old story or target of new therapeutic approaches?].

The complement system is a multifactorial protein cascade system which is essentially involved in the early unspecific immune response. Its major function is the activation of cellular defense mechanisms, opsonisation of foreign particles and the destruction of target cells. While the impact of the different complement components for bacterial elimination still remains controversial, overwhelming activation of the complement cascade, however, can induce life threatening tissue damage due to the effective cytotoxic properties. In the last years a variety of studies demonstrated beneficial, organ protective effects of complement modulation in models of severe inflammation. Attempts to control the complement system include the application of endogenous complement inhibitors e.g. C1-inhibitor (C1-INH) or the administration of recombinant complement receptors such as the soluble complement receptor 1 (rsCR1). Moreover antibodies against key proteins (C3, C5), against their activation products (C5a) or against complement receptor 3 (CR3, CD18/11b) mediated adhesion of leukocytes to the vascular endothelium, represent effective options of complement modulation. Besides this, insertion of membrane bound human complement regulators (DAF- CD55, MCP- CD46 or CD59) into xenogenic donor organs has proven effectiveness to prevent xenograft rejection. The described interventions protected from severe organ damage in various animal models of sepsis, myocardial and intestinal ischaemia-reperfusion injury, ARDS, nephritis, and xenograft rejection. With respect to recent clinical data, complement inhibition could represent a useful therapeutic strategy to control overwhelming inflammation. Own experiments demonstrated protective effects of complement modulation with C1 INH and rsCR1 in a model of complement induced pulmonary injury. With respect to sufficient host defense, however, the use of complement inhibitors must be considered carefully.

Complement Inactivator Proteins↗

PubMed2000

FreeStyle: a small-volume electrochemical glucose sensor for home blood glucose testing.

FreeStyle accurately measures the concentration of glucose in a 300-nL volume of capillary blood obtained with significantly reduced pain from the forearm where the density of capillaries is much lower than in the fingertips. The readings are independent of temperature, of altitude (O2 pressure) and of hematocrit in the 0%-60% range. At their physiological levels, none of the often interfering constituents of blood affect the readings.

Blood Glucose↗

PubMed2000

Endothelin-1 impairs neutrophil respiratory burst and elimination of Escherichia coli in rabbits.

OBJECTIVE: During systemic inflammation, elevated levels of endothelin (ET)-1 have been reported. The aim of this study was to investigate the effects of ET-1 on neutrophil (PMN) respiratory burst, phagocytosis, and elimination of Escherichia coli from blood and tissues. DESIGN: Prospective, randomized, controlled trial. SETTING: Experimental laboratory in a university hospital. SUBJECTS: A total of 18 female chinchilla rabbits. INTERVENTIONS: To quantify the clearance process, defined numbers (10(8) colony-forming units) of E. coli were injected intravenously into anesthetized rabbits, 60 mins after onset of continuous 0.2 microg/kg/min ET-1 administration (n = 9) and after saline infusion (control group, n = 9), respectively. To evaluate potential effects of ET-1 on bacterial elimination and killing, blood clearance of E. coli and colonization of different organs were investigated. MEASUREMENTS: Variables monitored were neutrophil respiratory burst and phagocytosis activity, rates of bacterial elimination from the blood, arterial blood pressure, blood gases, serum lactate concentrations, and nitrite and nitrate levels. The animals were killed 3 hrs after bacterial injection and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. MAIN RESULTS: Compared with the control group, ET-1 significantly impaired PMN respiratory burst (p < .05) and prolonged elimination of injected E. coli from the blood (p < .01), whereas phagocytosis functions remained unaltered. The reduced PMN burst activity after ET-1 was associated with a higher bacterial colonization of all organs (lung, p < .01; spleen, p < .05). Endothelin-1 induced increases in mean arterial pressure (p < .01) and serum lactate concentrations, whereas nitrite and nitrate levels remained unaltered. CONCLUSION: Endothelin-1 impairs respiratory burst and bacterial clearance from the blood and tissue. Thus, elevated levels of ET-1 during sepsis could induce organ hypoperfusion and cause disturbances in immune functions, increasing the risk of bacterial infections.

Animals↗

PubMed2000

Impact of endothelin-1 in endotoxin-induced pulmonary vascular reactions.

OBJECTIVES: Elevated endothelin-1 (ET-1) levels have been detected during sepsis. The aim of the study was to examine the role of thromboxane A2 (TXA2) and ET-1 in pulmonary vascular reactions after endotoxin (LPS) challenge. DESIGN: Prospective experimental study in rabbits. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Twenty-four adult rabbits of either sex. INTERVENTIONS: Experiments were performed on 30 isolated and ventilated rabbit lungs, which were perfused with a saline solution containing 10% autologous blood. MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial pressure and lung weight gain were continuously registered. Perfusate samples were drawn intermittently to determine ET-1, TXA2, and prostacyclin (PGI2) concentrations. LPS isolated from Escherichia coli (0.5 mg/mL; n = 6) was added to the perfusate. A marked pulmonary arterial pressure increase followed by massive edema formation after 60 mins was observed after LPS injection. At the same time, elevated TXA2 and PGI2 levels in the perfusate were measured. ET-1 was detected 30 mins after LPS infusion (13.4+/-2.6 fmol/L). Pretreatment with the ET(A) receptor antagonist LU135252 (10(-6) M; n = 6) almost completely suppressed the pressure reaction after endotoxin injection (p < .01 at 50 and 60 mins) and reduced edema formation (p < .05). The cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) was as effective as LU135252 in preventing vascular reactions after LPS injection. CONCLUSIONS: Pretreatment with the ET(A) receptor antagonist LU135252 and the cyclooxygenase inhibitor diclofenac reduced pulmonary vascular reactions after LPS challenge. Based on the current data, we conclude that the pulmonary arterial pressure increase and edema formation after LPS injection are related to an ET-1- and TXA2-dependent mechanism.

Animals↗

PubMed2000

The complement regulators C1 inhibitor and soluble complement receptor 1 attenuate acute lung injury in rabbits.

Because activation of the complement system plays a major role in the pathogenesis of acute lung injury, the availability of new specific complement inhibitors represents a promising therapeutic approach. In the present study we investigated pulmonary edema formation and pulmonary artery pressure (PAP) in acute complement-induced lung injury for possible therapeutic impact of the complement regulators C1 inhibitor and soluble complement receptor 1. Eighteen isolated and ventilated rabbit lungs were perfused with pooled normal human serum (NHS, final concentration 35%) in Krebs-Henseleit buffer in a recirculating system. Lung weight gain and PAP were continuously recorded. Complement activation was blocked by the addition of C1 inhibitor (1.0 U/mL, n = 6) or sCR 1 (2.0 microg/mL, n = 6). Lungs that received NHS without inhibitors served as controls (n = 6). This study was performed according to the Helsinki Declaration and approved by the local government. Application of NHS resulted in an increase of PAP within 20 min from 8+/-2 to 42+/-6 mmHg, which was significantly (P < 0.05) decreased by C1-Inh (25+/-5 mmHg) and sCRI (20 +/-3 mmHg). Moreover, pulmonary edema formation after NHS, as assessed by overall weight gain, was reduced by both C1-Inh and sCR1, compared with controls. These findings were paralleled with significantly decreased thromboxane release rates and reduced tissue deposition of C3c and C5b-9. C1 inhibitor and sCR1 attenuate the complement-induced pulmonary capillary leakage and PAP increase, indicating the protective effect of complement inhibition in isolated perfused rabbit lungs.

Animals↗

PubMed2000

Molecular cytogenetic characterisation of partial trisomy 9q in a case with pyloric stenosis and a review.

Partial trisomy 9q represents a rare and heterogeneous group of chromosomal aberrations characterised by various clinical features including pyloric stenosis. Here, we describe the case of a 1 year old female patient with different dysmorphic features including pyloric stenosis and prenatally detected partial trisomy 9q. This partial trisomy 9q has been analysed in detail to determine the size of the duplication and to characterise the chromosomal breakpoints. According to the data gained by different molecular cytogenetic techniques, such as fluorescence in situ hybridisation (FISH) with whole and partial chromosome painting probes, yeast artificial chromosome (YAC) probes, and comparative genomic hybridisation (CGH), the derivative chromosome 9 can be described as dup(9)(pter-->q22. 1::q31.1-->q22.1::q31.1--> q22.1::q31.1-->qter). Four breakpoint spanning YACs have been identified (y806f02, y906g6, y945f5, and y747b3) for the proximal breakpoint. According to this new case and previously published data, the recently postulated putative critical region for pyloric stenosis can be narrowed down to the subbands 9q22.1-q31.1 and is the result of either partial trisomy of gene(s) located in this region or a gene disrupted in 9q31.

Chromosome Banding↗

PubMed2000

Insulin-like growth factor serum concentrations reflect insufficient growth in a hypoplastic infant with partial trisomy 9q in the 12th week of life.

This report presents changes of IGFs and IGFBPs in a female infant with partial trisomy 9q in the 12th week of life. Studying deficient growth in this hypoplastic infant (birth weight 1405 g, birth length 36 cm) with dysmorphic features, the following changes in IGFs and IGFBPs were detected (microg/l): IGF-I: 26.5 vs 48.1 in healthy infants; IGF-II: 420 vs 728; IGFBP-2: 931 vs 524; IGFBP-3: 800 vs 1070. This demonstrates that IGFs and IGFBPs may reflect individual insufficient growth even at this early age.

Adult↗

PubMed2000

Microdissection based comparative genomic hybridization analysis (micro-CGH) of secondary acute myelogenous leukemias.

Comparative genomic hybridization (CGH) is a well established technique in molecular cytogenetics. However, leukemias, and especially secondary acute myelogenous leukemias (sAML) are not very well analyzed by this technique, even though such diseases are often characterized by complex karyotypic changes, not resolvable by conventional cytogenetic banding analysis. This lack of CGH-studies might be due to the fact, that in most cases bone marrow aspirate is too limited to do DNA-extraction additionally to the cytogenetic analysis. To circumvent this problem a new CGH technique has been applied to analyze 10 AML cases with complex karyotypic changes. In each case 15 interphase nuclei of the harvested and fixed bone marrow cell-suspension have been microdissected from the coverslip surface and collected in a tube. Subsequently, DNA was amplified by DOP-PCR. With this micro-CGH technique additional cytogenetic information from 10 highly aberrant AML cases was obtained and confirmed by FISH on metaphase of the corresponding AML case.

Aged↗

PubMed2000

[Immunonutrition with omega-3-fatty acids. Are new anti-inflammatory strategies in sight?].

In the early phase of sepsis and SIRS an overwhelming activation of humoral and cellular mediator systems can alter vascular resistance and causes capillary leakage increasing the risk of organ dysfunction. omega-6-arachidonic acid is released from lipid pools of cellular membranes during inflammation and is metabolized to pro-inflammatory prostaglandins and leukotriens, which are key mediators in the pathogenesis of organ dysfunction. omega-3-eicosapentaenoic acid-derived lipid mediators present altered biologic effects. Thus, omega-3-fatty acid application enables anti-inflammatory intervention on the level of lipid mediators. The current article reviews experimental and clinical data on omega-3-fatty acids. Besides the decrease of pro-inflammatory mediators, fish oil supplementation lowered post operative infection rates and showed a tendency to reduce hospital stay in surgical patients. It is believed that the decreased formation of LTB4 and TXA2 during sepsis after administration of omega-3-fatty acids accounts for improved microcirculatory perfusion and declined lactate acidosis.

Animals↗

PubMed1999

Antibody responses in pregnancy-induced transmammary transmission of Ancylostoma caninum hookworm larvae.

Third stage larvae of the Ancylostoma caninum hookworm nematode have the capacity to infect a dog, abort the normal maturation pathway to become blood-feeding intestinal worms, and instead distribute throughout the body in a developmentally arrested state that is relatively resilient to most chemotherapeutic agents. During pregnancy, a percentage of the arrested larvae reactivate and transmit via the mammary glands to infect the nursing puppies with resulting iron-deficiency anemia and potential mortality. To determine if the suppression of parasite-specific antibody responses during pregnancy facilitates the reactivation and transmammary transfer of hookworm larvae, a murine model of A. caninum infection was used to compare the infected versus uninfected animals that were either bred or not bred. Initial comparisons of genetically divergent BALB/c versus C57BL/6 mice showed that both the strains mounted strong Th2 biased IgG1 and IgE antibody responses to A. caninum infection. Using the BALB/c strain for the breeding analyses, it was confirmed that larval transfer to the mouse pups only occurred during the post-partum lactational period. In the dams, levels of total and antigen-specific IgG1 and total IgE were highly correlated with parasite burden. During most phases of pregnancy and lactation, infected dams had lower total IgG1, IgG2a and IgE levels as compared to unbred mice at comparable times post-infection; this downward modulation of antibody responses supports the established dogma of a generalized immunosuppression associated with pregnancy. However, at parturition and post-partum lactation, antigen-specific IgG1 levels measured at 1:5000 serum dilutions were comparable between bred and unbred mice, and antigen-specific IgG2a levels at 1:100 serum dilutions were also not significantly different except for a marginal reduction in the bred mice at the lactational timepoint. The comparable anti-A. caninum IgG1 levels between bred and unbred mice, and low correlation between IgG2a levels and larval burden suggest that parasite-specific antibody responses do not play a major role in the pregnancy-associated transmammary transmission of A. caninum larvae. This conclusion does not rule out the possibility that underlying fluxes in the levels of specific cytokines associated with pregnancy and infection may be involved in the process of larval reactivation and transmission.

Ancylostoma↗

PubMed1999

Rapid amperometric verification of PCR amplification of DNA.

Amplification of an 800-base template was verified in a 10-min test on a 2-microL sample of the PCR product solution. For verification, digoxigeninylated primers and biotinylated d-UTP-16-biotin were added to the amplification solution. The resulting amplified product was digoxigeninlabeled at its 3'-end and was also labeled with multiple biotin functions along its chain. The detecting electrode was coated with an electron-conducting redox hydrogel to which anti-digoxin monoclonal antibody was covalently bound. The amplified DNA was captured by the electrode through conjugation of its 3'-digoxigenin with the antibody. Exposure to a solution of horseradish peroxidase-labeled avidin led to capture of the enzyme and switched the redox hydrogel from a noncatalyst to catalyst for H2O2 electroreduction. The switching resulted in an H2O2 electroreduction current density of 2.1 +/- 0.9 microA cm-2 in 10-4 M H2O2 at Ag/AgCl potential and at 25 degrees C.

DNA↗

PubMed1999

Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition.

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.

Animals↗

PubMed1999

Forskolin and dopamine D1 receptor activation increase huntingtin's association with endosomes in immortalized neuronal cells of striatal origin.

Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntington's disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.

Animals↗