The ovotestis of Aplysia californica: anatomy and egg release

1980 ◽  
Vol 58 (12) ◽  
pp. 2220-2229 ◽  
Author(s):  
F. Edward Dudek ◽  
Hampik S. Injeyan ◽  
Bonnie Soutar ◽  
Greg Weir ◽  
Stephen S. Tobe
Keyword(s):  
Time Course ◽  
Aplysia Californica ◽  
Cell Extract ◽  
Continuous Exposure ◽  
Gradual Reduction ◽  
Release Process ◽  
Cell Extracts ◽  
High K ◽  
Scanning Electron

Egg release from the ovotestis of Aplysia californica has been studied using ovotestis fragments and bag cell extracts. Light and scanning electron microscopy showed clusters of follicles surrounded by muscle cells. Mature oocytes observed in egg masses and those released from ovotestis fragments were 90 μm in diameter. The number of mature releasable oocytes was relatively constant throughout the ovotestis, although a gradual reduction occurred with increasing distance from the small hermaphroditic duct.Bag cell induced egg release was detectable in vitro within 30 min and was complete by 180 min. The time course of egg release was similar under conditions of either continuous exposure or a 30-min pulse of bag cell extract. Artificial seawater (ASW) solutions with high K+ (110 mM) did not stimulate egg release unless bag cell extract was present. ASW with no Ca2+ and 3 mM EGTA or ASW containing Co2+ (10 mM) inhibited both bag cell induced and spontaneous (ASW alone) egg release.Therefore, brief exposure to bag cell peptide can trigger the egg release process, which is long lasting (~ 3 h) and Ca2+ dependent. The observation that high K+ did not stimulate egg release challenges the muscle contraction hypothesis of egg release.

Nitrogenase inactivation by oxygen and enzyme turnover in Anabaena cylindrica

1983 ◽  
Vol 29 (10) ◽  
pp. 1286-1294 ◽  
Author(s):  
Marcia A. Murry ◽  
Patrick C. Hallenbeck ◽  
Diane Esteva ◽  
John R. Benemann
Keyword(s):  
Time Course ◽  
Acetylene Reduction Activity ◽  
Anabaena Cylindrica ◽  
Reactive Material ◽  
Chloromethyl Ketone ◽  
Aerobic Conditions ◽  
Reduction Activity ◽  
Cell Extracts

Nitrogenase is known to be irreversibly inactivated by oxygen in vivo and in vitro. In time-course experiments using Anabaena cylindrica, cultures treated with antibiotics and incubated under various O2 tensions, in vivo acetylene reduction activity and immunologically determined Fe–Mo protein (component I) were lost at rates directly related to O2 tension. Activity was lost at a faster rate than cross-reactive material. The half-life of cross-reactive material was 25 h under microaerophilic conditions, 12 h under aerobic conditions, and 6.6 h under an O2 tension of 245% of air saturation. In vitro, cross-reactive material was lost in O2 exposed, but not in anaerobically prepared, crude cell extracts. Loss of cross-reactive material was prevented by freezing and by α-N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of histidine-residue proteases. These results indicate that nitrogenase is continuously inactivated by O2, hydrolyzed, and resynthesized during growth of this heterocystous cyanobacteria under aerobic conditions.

scholarly journals In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase

2000 ◽  
Vol 81 (8) ◽  
pp. 2095-2102 ◽  
Author(s):  
Yasuhiko Matsushita ◽  
Kohtaro Hanazawa ◽  
Kuniaki Yoshioka ◽  
Taichi Oguchi ◽  
Shigeki Kawakami ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Movement Protein ◽  
Cell Extract ◽  
Host Protein ◽  
Tomato Mosaic Virus ◽  
Stable Complex ◽  
Culture Cells ◽  
Cell Extracts

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [γ-32P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [γ-32P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5998-6006
Author(s):  
J R Patton
Keyword(s):  
Time Course ◽  
Buoyant Density ◽  
Early Time ◽  
Creatine Phosphate ◽  
Stem Loop ◽  
Cell Extracts ◽  
Nuclear Extracts ◽  
Binding Sequence ◽  
Made In

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.

scholarly journals Biosynthesis of the Egg-Laying Hormone (ELH) in the Bag Cell Neurons of Aplysia californica

1972 ◽  
Vol 60 (1) ◽  
pp. 102-119 ◽  
Author(s):  
S. Arch
Keyword(s):  
Molecular Weight ◽  
Aplysia Californica ◽  
Sodium Dodecyl ◽  
Tissue Perfusion ◽  
Egg Laying ◽  
Lower Molecular Weight ◽  
High K ◽  
Bag Cells ◽  
Bag Cell Neurons

Biosynthesis of the egg-laying hormone in the bag cell neurons of Aplysia californica was studied. Bag cells were incubated with leucine-3H in vitro for 30 min and rinsed for variable periods of time in a chase medium. The distribution of incorporated label among proteins within the cells was assayed by electrophoresis of an homogenate on sodium dodecyl sulfate polyacrylamide gels. Results from rinse times shorter than 30 min revealed that the predominant synthetic product is a 25,000 dalton protein. With longer rinse times, this species was reduced and two species of lower molecular weight became prominent. This redistribution of radioactivity was quantitative and was not prevented by inhibition of protein synthesis during the rinse. A 10°C reduction in temperature (from 15°C) blocked the redistribution. These data are interpreted to indicate that the 25,000 dalton molecule is a precursor which is cleaved enzymatically to yield two lower molecular weight products. One product is a 12,000 dalton molecule which remains in the cell bodies. The other is a molecule of <10,000 daltons which is exported from the somata into the neurohemal regions of the connective tissue. Perfusion of these regions with high [K+] medium results in the release of this product into the medium. It is concluded that this product is the 6000 dalton egg-laying hormone (ELH).

scholarly journals Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2

1999 ◽  
Vol 19 (7) ◽  
pp. 4897-4906 ◽  
Author(s):  
Que Lan ◽  
Kiyoshi Hiruma ◽  
Xiao Hu ◽  
Marek Jindra ◽  
Lynn M. Riddiford
Keyword(s):  
Cell Extract ◽  
Nuclear Hormone Receptor ◽  
Orphan Receptor ◽  
Early Gene ◽  
Upstream Region ◽  
Ecdysone Receptor ◽  
Mobility Shift ◽  
Receptor Binding Site ◽  
Cell Extracts

ABSTRACT MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5′ flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 μg of 20E per ml inManduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1–USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at −671 and the adjacent GGGTCA being most critical. The EcRE1 at −342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1–USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1–USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1–USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1–USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the sameKd (7 × 10−10 M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.

scholarly journals Production of cartilage link protein by human granulosa-lutein cells

2002 ◽  
Vol 175 (2) ◽  
pp. 505-515 ◽  
Author(s):  
GW Sun ◽  
H Kobayashi ◽  
M Suzuki ◽  
N Kanayama ◽  
T Terao
Keyword(s):  
Time Course ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Extracellular Matrix Protein ◽  
Regulatory Function ◽  
Human Ovary ◽  
Vitro Fertilization ◽  
Link Protein ◽  
Cell Extracts

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

Functional characterization of the putative FAD synthase from Mycoplasma hyopneumoniae

2021 ◽  
Vol 368 (3) ◽  
Author(s):  
Amanda Malvessi Cattani ◽  
Camila Vieira Pinheiro ◽  
Irene Silveira Schrank ◽  
Franciele Maboni Siqueira
Keyword(s):  
Drug Targets ◽  
Functional Characterization ◽  
3D Structure ◽  
Mycoplasma Hyopneumoniae ◽  
Cell Extract ◽  
Enzymatic Assays ◽  
Cell Extracts ◽  
Potential Drug Targets

ABSTRACT In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.

Intracellular Ca2+ stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

2000 ◽  
Vol 279 (1) ◽  
pp. C51-C61 ◽  
Author(s):  
I. Vicario ◽  
A. Obeso ◽  
A. Rocher ◽  
J. R. López-Lopez ◽  
C. González
Keyword(s):  
Significant Role ◽  
Carotid Body ◽  
Time Course ◽  
Cyclopiazonic Acid ◽  
Structural Identity ◽  
High K ◽  
Intracellular Ca ◽  
Intact Rabbit ◽  
Chemoreceptor Cells

The notion that intracellular Ca2+ (Cai 2+) stores play a significant role in the chemoreception process in chemoreceptor cells of the carotid body (CB) appears in the literature in a recurrent manner. However, the structural identity of the Ca2+ stores and their real significance in the function of chemoreceptor cells are unknown. To assess the functional significance of Cai 2+ stores in chemoreceptor cells, we have monitored 1) the release of catecholamines (CA) from the cells using an in vitro preparation of intact rabbit CB and 2) the intracellular Ca2+ concentration ([Ca2+]i) using isolated chemoreceptor cells; both parameters were measured in the absence or the presence of agents interfering with the storage of Ca2+. We found that threshold [Ca2+]i for high extracellular K+ (Ke +) to elicit a release response is ≈250 nM. Caffeine (10–40 mM), ryanodine (0.5 μM), thapsigargin (0.05–1 μM), and cyclopiazonic acid (10 μM) did not alter the basal or the stimulus (hypoxia, high Ke +)-induced release of CA. The same agents produced Cai 2+transients of amplitude below secretory threshold; ryanodine (0.5 μM), thapsigargin (1 μM), and cyclopiazonic acid (10 μM) did not alter the magnitude or time course of the Cai 2+responses elicited by high Ke +. Several potential activators of the phospholipase C system (bethanechol, ATP, and bradykinin), and thereby of inositol 1,4,5-trisphosphate receptors, produced minimal or no changes in [Ca2+]i and did not affect the basal release of CA. It is concluded that, in the rabbit CB chemoreceptor cells, Cai 2+ stores do not play a significant role in the instant-to-instant chemoreception process.

scholarly journals Experimental and Modeling Studies of Novel Bursts Induced by Blocking Na+ Pump and Synaptic Inhibition in the Rat Spinal Cord

2002 ◽  
Vol 88 (2) ◽  
pp. 676-691 ◽  
Author(s):  
Aldo Rozzo ◽  
Laura Ballerini ◽  
Gilda Abbate ◽  
Andrea Nistri
Keyword(s):  
Spinal Cord ◽  
Time Course ◽  
Synaptic Depression ◽  
Neonatal Rat ◽  
Pump Operation ◽  
High K ◽  
Close Sequence ◽  
Activity Dependent ◽  
Dependent Processes

This study addressed some electrophysiological mechanisms enabling neonatal rat spinal networks in vitro to generate spontaneous rhythmicity. Networks, made up by excitatory connections only after block of GABAergic and glycinergic transmission, develop regular bursting (disinhibited bursts) suppressed by the Na+ pump blocker strophanthidin. Thus the Na+ pump is considered important to control bursts. This study, however, shows that, after about 1 h in strophanthidin solution, networks of the rat isolated spinal cord surprisingly resumed spontaneous bursting (“strophanthidin bursting”), which consisted of slow depolarizations with repeated oscillations. This pattern, recorded from lumbar ventral roots, was synchronous on both sides, of irregular periodicity, and lasted for ≥12 h. Assays of 86Rb+uptake by spinal tissue confirmed Na+ pump block by strophanthidin. The strophanthidin rhythm was abolished by glutamate receptor antagonists or tetrodotoxin, indicating its network origin. N-methyl-d-aspartate (NMDA), serotonin, or high K+ could not accelerate it. The size of each burst was linearly related to the length of the preceding pause. Bursts could also be generated by dorsal root electrical stimulation and possessed similar dependence on the preceding pause. Conversely, disinhibited bursts could be evoked at short intervals from the preceding one unless repeated pulses were applied in close sequence. These data suggest that rhythmicity expressed by excitatory spinal networks could be controlled by Na+ pump activity or slow synaptic depression. A model based on the differential time course of pump operation and synaptic depression could simulate disinhibited and strophanthidin bursting, indicating two fundamental, activity-dependent processes for regulating network discharge.

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

1983 ◽  
Vol 61 (9) ◽  
pp. 1025-1034 ◽  
Author(s):  
C. W. Loomis ◽  
J. F. Brien
Keyword(s):  
Oral Administration ◽  
Time Course ◽  
Time Interval ◽  
Maximum Plasma ◽  
Control Activity ◽  
Calcium Cyanamide ◽  
High K ◽  
Low K

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.

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