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B Hacker

Publications and source records attributed to B Hacker.

At least 19 recordsLinked to original sources
PubMed2000

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.

Cyclic nucleotide-gated ion channels in olfactory sensory neurons (OSNs) are hypothesized to play a critical role in olfaction. However, it has not been demonstrated that the cAMP signaling is required for olfactory-based behavioral responses, and the contributions of specific adenylyl cyclases to olfaction have not been defined. Here, we report the presence of adenylyl cyclases 2, 3, and 4 in olfactory cilia. To evaluate the role of AC3 in olfactory responses, we disrupted the gene for AC3 in mice. Interestingly, electroolfactogram (EOG) responses stimulated by either cAMP- or inositol 1,4,5-triphosphate- (IP3-) inducing odorants were completely ablated in AC3 mutants, despite the presence of AC2 and AC4 in olfactory cilia. Furthermore, AC3 mutants failed several olfaction-based behavioral tests, indicating that AC3 and cAMP signaling are critical for olfactory-dependent behavior.

Adenylyl Cyclases↗
PubMed1998

Impaired cerebellar long-term potentiation in type I adenylyl cyclase mutant mice.

Activation of adenylyl cyclase and the consequent production of cAMP is a process that has been shown to be central to invertebrate model systems of information storage. In the vertebrate brain, it has been suggested that a presynaptic cascade involving Ca influx, cAMP production, and subsequent activation of cAMP-dependent protein kinase is necessary for induction of long-term potentiation (LTP) at the cerebellar parallel fiber-Purkinje cell synapse. We have used mutant mice in which the major Ca-sensitive adenylyl cyclase isoform of cerebellar cortex (type I) is deleted to show that this results in an approximately 65% reduction in cerebellar Ca-sensitive cyclase activity and a nearly complete blockade of cerebellar LTP assessed using granule cell-Purkinje cell pairs in culture. This blockade is not accompanied by alterations in a number of basal electrophysiological parameters and may be bypassed by application of an exogenous cAMP analog, suggesting that it results specifically from deletion of the type I adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1997

Sensitization of olfactory guanylyl cyclase to a specific imprinted odorant in coho salmon.

The role of cGMP in olfactory signaling is not fully understood, but it is believed to play a modulatory role in intracellular signaling in vertebrate olfactory receptor neurons (ORNs). Here, we present evidence that cGMP in ORNs may play an important role in recognition of biologically relevant odors and olfactory learning. Specifically, we investigated the cellular mechanisms underlying olfactory imprinting in salmon. Salmon learn odors associated with their natal site as juveniles and later use these odors to guide their homing migration. This imprinting is believed to involve sensitization of the peripheral olfactory system to specific homestream odorants. We imprinted juvenile salmon to the odorant beta-phenylethyl alcohol (PEA) and examined the sensitivity of olfactory adenylyl and guanylyl cyclases to PEA during development. Stimulation of guanylyl cyclase activity by PEA was significantly greater in olfactory cilia isolated from PEA-imprinted salmon compared with PEA-naive fish only at the time of the homing migration, 2 years after PEA exposure. These results suggest that sensitization of olfactory guanylyl cyclase may play an important role in olfactory imprinting by salmon.

Animals↗
PubMed1994

Site-directed mutagenesis of arginine-114 and tryptophan-129 in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides: two highly conserved residues predicted to be near the cytoplasmic surface of putative transmembrane helices B and C.

The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.

Amino Acid Sequence↗
PubMed1993

Characterization of mutations in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides that affect the quinone reductase site (Qc).

The cytochrome b subunit of the bc1 complex contains two heme components, cytochrome bL and cytochrome bH, and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc). The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO. In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis. These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Qc-directed inhibitors. The site-directed mutants all exhibit a normal rate of reduction of cytochrome bH, suggesting a fully functional quinol oxidizing site. However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome bH. Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc1 complex. In both cases, the cause of the defect is the lack of reoxidation of cytochrome bH by ubiquinone. This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome bH to the Qc-site. This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.

Amino Acid Sequence↗
PubMed1993

The bc1 complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus.

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochrome c oxidoreductase (bc1 complex). In both Rhodobacter sphaeroides and Rhodobacter capsulatus, the bc1 complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because the bc1 complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochrome b subunit, in the Rieske iron-sulfur subunit, and in the cytochrome c1 subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Qo) site or the quinol reductase (Qi) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.

Amino Acid Sequence↗
PubMed1992

Structure and function of the bc-complex of Rhodobacter sphaeroides.

The ubiquinol:cytochrome c2 oxidoreductase (bc-complex) of Rhodobacter sphaeroides has three main subunits, which bear the prosthetic groups, and contribute to three catalytic sites and internal electron transfer pathways which define the modified Q-cycle mechanism. In this paper, we report on progress in modelling the structure of the bc-complex, and experiments using site directed mutagenesis and biophysical assay to probe the structural and function consequences of specific modifications to these subunits.

Benzoquinones↗
PubMed1984

Cytotoxic effects of Eleutherococcus senticosus aqueous extracts in combination with N6-(delta 2-isopentenyl)-adenosine and 1-beta-D-arabinofuranosylcytosine against L1210 leukemia cells.

The use of the aqueous extracts of Eleutherococcus senticosus in combination with either cytarabine or N6-(delta 2-isopentenyl)-adenosine gave additive antiproliferative effects against L1210 murine leukemia. The ED50 for E. senticosus root extracts against L1210 cells was approximately 75 micrograms/mL. E. senticosus appears to be potentially useful for reducing the concentration of conventional antimetabolites used for their antiproliferative effects on tumor cells.

Adenine↗
PubMed1983

Enhancement of the antitumor activity of N6-(delta 2-isopentenyl)adenosine against cultured L-1210 leukemia cells by pentostatin using a polymeric delivery system.

The adenosine deaminase inhibitor pentostatin (I), recently shown to be effective in the treatment of several types of acute and chronic human leukemias, was impregnated in a silicone polymer monolithic disk device for release in vitro in the presence of the antitumor nucleoside N6-(delta 2-isopentenyl)adenosine (II) against mouse L-1210 lymphocytic leukemia cells. Although I is ineffective alone against L-1210 cells, controlled release from the polymeric delivery matrix potentiates the antiproliferative effects of II during the midlog phase of growth (48 hr). Cytotoxicity is prolonged, leading to total cell death during the stationary phase of growth (96 hr). The present study suggests that polymeric delivery systems be used for controlled release of oncologic agents, alone or in combination with inhibitors, especially where liability is a concern.

Adenosine↗
PubMed1983

Increased cytotoxicity of N6-(delta 2-isopentenyl)adenosine in combination with pentostatin against L-1210 leukemia cells.

Pentostatin (I), a tight-binding inhibitor of adenosine deaminase, was evaluated in combination with the partially effective antitumor nucleoside N6-(delta 2-isopentenyl)adenosine (II) for cytotoxic activity against cultured L-1210 lymphocytic mouse leukemia cells. Although I alone (less than or equal to 10 micrograms/ml) was ineffective, it significantly potentiated and prolonged the cytotoxic and cytostatic activities of II. The combination of I (2-10 micrograms/ml) with II (25 micrograms/ml) resulted in inhibition of cellular proliferation (80-96%) within 24 hr with maintenance at that level for an extended period of time due to the continued ability of I to prevent the facile deamination of the allylic side chain of II. This type of adjuvant chemoprotection has potential use for other labile oncologic agents.

Adenosine↗
PubMed1982

Antineoplastic effects of N6-(delta 2-isopentenyl)adenosine against L-1210 mouse lymphocytic leukemic cells using a polymeric delivery system.

N6-(delta 2-Isopentenyl)adenosine (I), a nucleoside previously shown to be cytotoxic against several types of tumor cells, was impregnated in silicone polymer monolithic disc devices for release in vitro against lymphocytic mouse leukemia cells. Plotting the cumulative amount of N6-(delta 2-isopentenyl)adenosine released per unit area of the device versus the square root of time revealed a linear relationship. However, the higher loading dose tended to rapidly release any drug deposited on the polymer surface. The optimum loading dose of the device for the most effective antileukemic activity in 24 hr was calculated based on a plot of the release rate versus the square root of an initial loading dose. The silicone polymer--I delivery system enabled a sustained and controllable release of additional agent. It was thus possible to achieve virtually total inhibition of leukemia cell replication using the polymeric delivery system. Increased concentrations of I, without the use of the polymeric system, resulted in maximum 24 hr inhibition of only approximately 81%, followed by a decline in overall antileukemic activity. It is possible to achieve a more predictable release rate of N6-(delta 2-isopentenyl)adenosine and corresponding antileukemic activity using a polymeric delivery system against L-1210 mouse leukemic cells in vitro. The relative data indicate the ED50 concentrations to be considerably less using the polymeric delivery system.

Adenosine↗
PubMed1982

Comparison of prostatic and nonprostatic acid phosphatase.

Electrophoresis and ion-exchange column chromatography were used to separate the wide varieties of acid phosphatases with different biological and clinical significance. Band 0 was very strong in ascitic cells with many autophagic vacuoles, indicating a role in autophagic function. Band 1 was a membrane-bound acid phosphatase, seen mainly in the microsomal fraction. Band 3 was the major lysosomal acid phosphatase of all nonprostatic tissues. Bands 2 and 4 were antigenically identical to each other, and were observed in unusually high amounts in the prostate. The different electrophoretic mobility between bands 2 and 4 was due to their carbohydrate content. Band 5 was a characteristic enzyme of the osteoclast. The tartrate-sensitive enzymes included bands 0 through 4. Only band 5 was tartrate resistant. The tartrate-resistant acid phosphatase of erythrocytes was not detected by the electrophoresis method. Clinical applications were seen for both bands 2 and 5. Band 2 was a secretory enzyme, normally secreted into the seminal plasma. Band 2 was absorbed into the blood circulation in some prostatic cancer patients. A small amount of bands 2 and 4 was observed in nonprostatic tissues. The diagnostic value of band 2 resulted from its extremely high concentration in the prostate. Band 5 was not observed in the normal prostate. A high concentration of band 5 was observed in hairy cells, Gaucher cells, and osteoclasts. The serum level of band 5b was an indicator of osteoclastic activity in the bone. Elevation of band 5b in serum was observed in normal children during physiological bone growth, in Gaucher's disease, and in malignancies metastasized to bone.

Acid Phosphatase↗
PubMed1980

Single subject research strategies in occupational therapy, part 1.

This paper is designed to orient occupational therapists to the use of single subject methodology as a viable mode of clinical research. The role of single subject designs in today's scientific world is explored through an historical perspective, and major strengths and weaknesses are discussed. Basic methodological concepts and research designs are presented to provide a foundation for therapists interested in documenting intervention results. Finally, some of the problems inherent in transferring single subject strategies to occupational therapy are presented.

History, 19th Century↗
PubMed1980

Single subject research strategies in occupational therapy: part 2.

This second of a 2-part paper on single subject research is devoted to practical implementation issues. Using clinical examples, the challenge of transferring single subject research strategies to occupational therapy intervention approaches is explored. The therapeutic approaches of behavioral, motoric, and processing are employed to discuss the use of single subject methodology in occupational therapy. Initial research activities for those interested in documenting treatment results are described. Finally, directions for the future use of single subject research are discussed.

Behavior Therapy↗
PubMed1975

Biochemical profiles of cancer cells: I. Computerized analysis of mouse leukemic cellular RNA on polyacrylamide gels.

Recent studies in this laboratory have been directed at investigating the cellular and subcellular metabolism of RNA in leukemic cells which have been characterized with respect to their degree and type of sensitivity/resistance to specific chemotherapeutic agents. In the present report, electrophoretic patterns on several types of SDS-polyacrylamide gels are presented using total cellular RNA preparations from a subline of L1210 mouse leukemia found to be resistant to cytosine arabinoside (L1210/Ara-C). These studies have been facilitated by using a computerized-spectrophotometric system for quantitative and qualitative comparisons of these profiles. The results suggest that patterns of RNA metabolism may be a useful biochemical test in leukemia.

Animals↗