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The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a key role in the control of eukaryotic cellular activity. The enzymology of PK-A in the free-living nematode, Caenorhabditis elegans is deceptively simple. Single genes encode the catalytic (C) subunit (kin-1), the regulatory (R) subunit (kin-2) and an A-kinase anchor protein (AKAP) (aka-1); nonetheless, PK-A is able to facilitate a comprehensive array of cAMP-mediated processes in this model multicellular organism. We have previously demonstrated that, in C. elegans, as many as 12 different isoforms of the C-subunit arise as a consequence of alternative splicing strategies. Here, we report the occurrence of transcripts encoding novel isoforms of the PK-A R-subunit in C. elegans. In place of exons 1 and 2, these transcripts include coding sequences from novel B or Q exons directly linked to exon 3, thereby generating isoforms with novel N‐termini. R-subunits containing an exon B-encoded N-terminal polypeptide sequence were detected in extracts prepared from mixed populations of C. elegans. Of note is the observation that R-subunit isoforms containing exon B- or exon Q-encoded polypeptide sequences lack the dimerisation/docking domains conventionally seen in R-subunits. This means that they are unlikely to participate in the formation of tetrameric PK-A holoenzymes and, additionally, they are unlikely to interact with AKAP(s). It is therefore possible that, in C. elegans, in addition to tetrameric (R2C2) PK-A holoenzymes, there is also a sub-population of dimeric (RC) PK-A enzymes that are not tethered by AKAPs. Furthermore, inspection of the N-terminal sequence encoded by exon B suggests that this isoform is a likely target for N-myristoylation. Although unusual, a number of similarly N-myristoylatable R-subunits, from a range of different species, are present in the databases, suggesting that this may be a more generally observed feature of R-subunit structure. The occurrence of R-subunit isoforms, without dimerisation/docking domains (with or without N-myristoylatable N-termini) in other species would suggest that the control of PK-A activity may be more complex than hitherto thought.

Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N′3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N′1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N′1 C-subunit isoforms, following incubation of nematodes with [3H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl–glycine in the C-subunit. In order to better understand the impact of the N′1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N′1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N′1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit.

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.

Recent discoveries of extensive mineral sand deposits in the eastern part of the Eucla Basin, South Australia, have generated much interest in their potential as a new world-class resource for heavy minerals. A detailed mineral characterisation study of a range of ilmenite concentrates from the Eucla Basin was undertaken using automated electron microprobe-based mineral mapping and quantitative analysis methods. Results showed that the ilmenite concentrates have a high bulk TiO2 content (>60 wt.%) consistent with a mineral assemblage dominated by the hydrated, Ti-rich alteration phase pseudorutile (Fe3+2−xTi3O9−3x(OH)3x). Minor accessory phases (

Administration of the nonsteroidal ecdysteroid agonist RH-5849 or 20-hydroxyecdysone to final larval instar tobacco hornworm, Manduca sexta, has been shown to induce an ecdysteroid inactivation enzyme, ecdysteroid 26-hydroxylase, and the cytosolic inactivation enzymes ecdysone oxidase and ecdysteroid phosphotransferase. In this work, we show that induction of ecdysteroid 26-hydroxylase by the ecdysteroid agonists RH-5849, RH-5992 (tebufenozide), and RH-0345 (halofenozide) appears universal in lepidopteran species that show susceptibility to the agonists. Interestingly, the waxmoth, Galleria mellonella, which shows very low susceptibility to the agonists but whose ecdysteroid receptor is capable of binding the agonist, shows no induction of a 26-hydroxylase activity. It appears that the more potent ecdysteroid agonists in Lepidoptera, RH-5992 and RH-0345, show in general a greater induction of 26-hydroxylase than RH-5849. Feeding RH-5849 to the dipteran housefly Musca domestica results in the induction of an ecdysteroid phosphotransferase. The low toxicity of these ecdysteroid agonists in orthopteran and coleopteran orders also correlates with a lack of induction of ecdysteroid 26-hydroxylase activity. We propose that in species where ecdysteroid agonists are effective in stimulating an untimely premature molt, a response to a state of hyperecdysonism elicited by the agonists is induction of enzymes of ecdysteroid inactivation.

Phase relations and the stability of crystallographic shear (CS) structure compounds (Cr,Fe)2Tin−2O2n−1 in the system Fe2O3–Cr2O3–TiO2 were investigated between 1000 and 1300°C. The ternary comprises five major solid solution series. These are as follows: an M2O3 series; an M3O5 series made up of two separate solid solution series—the first an orthorhombic pseudobrookite M3O5 solid solution and the second a monoclinic M3O5 series based on the V3O5 structure type; an M4O7 series; and an M5O9 series. These latter three series represent lower homologues (n=3, 4, and 5) of the (Cr,Fe)2Tin−2O2n−1CS compound series. Between adjacent M3O5 and M4O7 and M4O7 and M5O9 solid solutions, ordered intergrowths may occur. The stability and compositional limits of the solid solution series and intergrowth phases are dependent upon the temperature and Fe:Cr ratio. At high-TiO2 contents, assemblages may contain either members of the Andersson phase series Cr2Tin−2O2n−1, a continuous CS structure series extending into the ternary, or a rutile-based solid solution. A comparison of results from this study with previously published phase relations has led to a revised version of the Fe2O3–Cr2O3–TiO2 phase diagram.

Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.

Classical models of intracellular signalling describe how small changes in a cell's external environment can bring about major changes in cellular activity. Recent findings from experimental biology indicate that many intracellular signalling systems show a high level of spatial organisation. This permits the modification, by protein kinase or protein phosphatase action, of specific subsets of intracellular proteins - an attribute that is not addressed in classical signalling models. Here we use ideas and concepts from computer science to describe the information processing nature of intracellular signalling pathways and the impact of spatial heterogeneity of their components (e.g. protein kinases and protein phosphatases) on signalling activity. We argue that it is useful to view the signalling ecology as a vast parallel distributed processing network of agents operating in heterogeneous microenvironments, and we conclude with an overview of the mathematical and semantic methodologies that might help clarify this analogy between biological and computational systems.

In eucaryotic organisms, responses to external signals are mediated by a repertoire of intracellular signalling pathways that ultimately bring about the activation/inactivation of protein kinases and/or protein phosphatases. Until relatively recently, little thought had been given to the intracellular distribution of the components of these signalling pathways. However, experimental evidence from a diverse range of organisms indicates that rather than being freely distributed, many of the protein components of signalling cascades show a significant degree of spatial organisation. Here, we briefly review the roles of 'anchor' 'scaffold' and 'adaptor' proteins in the organisation and functioning of intracellular signalling pathways. We then consider some of the parallel distributed processing capacities of these adaptive systems. We focus on signalling proteins-both as individual 'devices' (agents) and as 'networks' (ecologies) of parallel processes. Signalling proteins are described as 'smart thermodynamic machines' which satisfy 'gluing' (functorial) roles in the information economy of the cell. This combines two information-processing views of signalling proteins. Individually, they show 'cognitive' capacities and collectively they integrate (cohere) cellular processes. We exploit these views by drawing comparisons between signalling proteins and verbs. This text/dialogical metaphor also helps refine our view of signalling proteins as context-sensitive information processing agents.

Diadenosine polyphosphates (Ap n As) can, through interaction with appropriate purinoceptors, affect a range of cellular activities. Ap 4 A, the most prominent naturally occurring diadenosine polyphosphate, stimulates alterations in intracellular calcium homeostasis and subsdquent activation of glycogen breakdown in isolated liver cells. Here we show that Ap 4 A, and other naturally occurring diadenosine polyphosphates, also stimulates phospholipase D (PLD) activity in isolated rat liver cells. The characteristics of Ap 4 A-mediated activation of PLD are similar to those for the activation of PLD by extracellular ATP. These results are discussed in the context of the relation between diadenosine polyphosphate– and adenine mononucleotide–mediated cellular signalling processes.

Neonates with congenital diaphragmatic hernia (CDH) have a high morbidity and mortality rate caused by pulmonary hypoplasia associated with pulmonary hypertension (PH). In experimental CDH, antenatal glucocorticoid therapy improves surfactant biochemical immaturity, enhances lung compliance, and induces morphological maturation in CDH rats. The effects of steroid therapy on preventing or treating PH in this condition have not been studied. Angiotensin converting enzyme (ACE), which is produced by the vascular endothelium, is implicated in the pathogenesis of pulmonary hypertension. The aim of this study was to evaluate the effect of antenatal glucocorticoid therapy on ACE activity and expression in CDH rat lungs.CDH was induced in fetal rats by the maternal administration of 100 mg nitrofen on day 9.5 of gestation (term, day 22). Dexamethasone (Dex) (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 before delivery of the fetuses by cesarean section on day 21.5. Control animals received olive oil (OO) by gavage and normal saline (NS) as vehicle injection. ACE activity was measured spectrophotometrically in the lungs of rats from four treatment groups: CDH-NS, non-CDH-NS, CDH-Dex, and OO-NS controls. Total lung ACE activity (mU per lung) was significantly lower in CDH-NS (P = .002) and CDH-Dex (P = .004) rats compared with non-CDH-NS and OO-NS controls (9.1 +/- 1.0 and 10.7 +/- 1.3 v 16.2 +/- 1.6 and 15.4 +/- 1.7). When specific ACE activity (mU/mg protein) was derived by expressing ACE activity per milligram of lung protein, CDH-NS animals showed elevated specific ACE activity (P = .05) compared with OO-NS controls (6.31 +/- 1.1 v 4.4 +/- 0.4). CDH-Dex animals had a significantly lower specific ACE activity (P = .01) compared with CDH-NS and Non-CDH-NS rats (4.0 +/- 0.4 v 6.31 +/- 1.1 and 5.83 +/- 0.54). The specific ACE activity levels of CDH-Dex rats were equivalent to those seen in the lungs of OO-NS controls (P = .24).Antenatal steroid therapy, by suppressing pulmonary ACE activity, may reduce the risk of pulmonary hypertension developing in human newborns with antenatally diagnosed CDH.

A rapid, highly-specific ELISA for tyrosine kinases readily detects such activity in crude extracts prepared from rat mammary epithelial and fibroblastic cells that have been stimulated with epidermal growth factor or basic fibroblast growth factor. Tyrosine kinase activity is also found in extracts of pupae of the cotton leafworm, Spodoptera littoralis. Both the mammalian and the insect tyrosine kinases are ATP-dependent. Both cytosol and membrane-associated (Triton-X-100-soluble) fractions of Spodoptera littoralis pupae contain tyrosine kinase activity. The growth factor-stimulated tyrosine kinase activities in extracts of growth factor-stimulated rat mammary cells are inhibited by genistein and an analogue of erbstatin: methyl 2,5-dihydroxycinnamate. However, the tyrosine kinase activities present in pupae of Spodoptera littoralis are not sensitive to these inhibitors, suggesting that the major tyrosine kinases of Spodoptera littoralis pupae may be distinct from the growth factor-stimulated mammalian tyrosine kinases.

The ability of purine nucleotides (e.g. ATP) to cause a dose-dependent activation of glycogen phosphorylase in isolated liver cells is well known known. These agents mediate their effects through interaction with specific P2-purinoceptors in the plasma membrane. We have investigated the ability of a range of synthetic and naturally occurring adenine dinucleotides to cause a similar stimulation of glycogen phosphorylase activity in isolated rat liver cells. Our results indicate that Ap3A and Ap4A, the most abundant naturally occurring adenine dinucleotides, cause a dose-dependent activation of glycogen phosphorylase similar to that observed with ATP. Similar responses were seen with Ap5A, Ap6A and a series of phosphorothioate analogues. In contrast, the response to phosphonate analogues depended on the position of the P-C-P bridged. The dinucleotides appear to exert their effects directly, rather than through hydrolytic products such as adenosine and/or ATP. The possibility that adenine dinucleotides are physiologically significant extracellular purinergic effectors is discussed in the light of these observations.

Electrical and mechanical correlates of crural diaphragm activity during swallow-induced and transient lower esophageal sphincter relaxation were monitored in 12 healthy subjects. Simultaneous esophageal manometric, pH, and crural diaphragm electromyogram recordings were performed for 1 hour in the postprandial period. Swallow-induced lower esophageal sphincter relaxation was associated with minimal inhibition of the crural diaphragm, but transient lower esophageal sphincter relaxation was accompanied by marked inhibition of the crural diaphragm. The degree of lower esophageal sphincter relaxation appeared to correlate with the degree of crural diaphragm inhibition during transient lower esophageal sphincter relaxation. Inhibition of crural diaphragm during transient lower esophageal sphincter relaxation may play an important role in facilitating flow across the gastroesophageal junction.

The total activity of three key enzymes and the flux through eight steps of aromatic amino acid metabolism have been determined in liver cells isolated from rats fed either control or pyridoxine-free diet for 5–6 weeks. The pyridoxine-free diet caused a decrease in the catabolism of tyrosine and phenylalanine because of a drop in the flux through tyrosine aminotransferase. This decrease of expressed cellular tyrosine aminotransferase activity can be fully explained in terms of loss of cofactor. Larger decreases in the catabolism of tryptophan were seen after pyridoxine deprivation. The decreased extent of tryptophan catabolism can be solely attributed to loss of cofactor or increased degradation of kynureninase. Inhibition of tryptophan 2,3dioxygenase was seen in pyridoxine deficiency, probably because of the buildup of the kynurenine metabolites. The control strength of kynureninase, for flux through kynureninase, was calculated to be less than or equal to 0.004, but 0.41 after pyridoxine deprivation. The sensitivity of the three pathways to pyridoxine deprivation is interpreted and discussed in terms of the different affinities for pyridoxal phosphate and the control strengths of the pyridoxal phosphate-dependent enzymes, tyrosine aminotransferase and kynureninase.

Upper Triassic continental strata in the United States contain rare horizons which yield pollen assemblages. The Dockum Group and Chinle Formation of the American Southwest, as well as certain rock units within the Deep River, Richmond, and Taylorsville Basins of the Newark Group, contain palynomorphs which exhibit stratigraphically restricted ranges in Europe. The presence of Vallasporites ignacii, Camerosporites secatus and Ovalipollis ovalis in the Dockum Group, Chinle Formation, Cumnock Formation of the Deep River Basin, Vinita Formation of the Richmond Basin, and unnamed units in the Taylorsville Basin suggest that all these units are approximately contemporaneous. The occurrence of these and other stratigraphically diagnostic pollen taxa, particularly Patinasporites densus, further indicates a Middle to Late Karnian age for these units. The palynostratigraphy also implies that the Pekin Formation of the Deep River Basin is slightly older than the above mentioned units.

A system for the parallel determination of enzyme phosphorylation and expressed activity in rat liver cells, and its application to studies of phenylalanine hydroxylase and tyrosine aminotransferase, is described. Phenylalanine hydroxylase is phosphorylated by agents which stimulate cyclic AMP- and Ca 2+ -dependent protein kinase activity. The phosphorylation site(s) appear to be the same for both kinases. Phosphorylation is accompanied by increased metabolic flux at low, physiologically relevant, substrate concentrations. Insulin and spermine both inhibit the phosphorylation of the enzyme, possibly by increasing dephosphorylation. Tyrosine aminotransferase is phosphorylated in liver cell incubations but the rate is slow and insensitive to additions to the medium. No parallel changes in flux could be detected. Both enzymes are subject to complex regulatory mechanisms, short- and long-term. Their activities may be coordinated in vivo by control exerted at the level of the plasma membrane where both amino acids share the same transport processes. Determination of the control coefficients for the several components indicates that membrane transport may be a major limitation on flux.

B. S., a white maIe age fifty, was admitted to the Maimonides HospitaI on December 4, 1947. He stated that about four hours prior to admission he stepped from his bath, went to stoo1, was frightened whiIe pIaying with his dog, causing him to faI1 from the toiIet seat onto a gIass of the type used for oId fashioned cocktaiIs which was on the floor nearby. The gIass entered the rectum. The patient was a we11 nourished individua1 in exceIIent physica condition but in obvious distress. BIood pressure, puIse and respirations were within norma Iimits. The bIadder was emptied of 1,000 cc. of cIear urine which upon microscopic examination was not unusua1. Prior to his admission the patient had taken a Iarge dose of castor oil and now compIained of severe abdomina1 cramps, tenderness in the Iower abdomen and moderate distention, Under spina anesthesia of 150 mg. of procaine the examining surgeon was abie to paIpate the rim of the gIass, but repeated attempts to remove it were unsuccessfu1, severa pieces being broken, resuIting in Iacerations of the recta1 mucosa. Packing was inserted to check hemorrhage. On the foIIowing day because of severe abdomina pain and marked abdomina1 distention, a coIostomy was performed through a Ieft Iower rectus muscIe spIitting incision, using a spina anesthetic of 150 mg. of procaine. The gIass couId be paIpated through the abdomen but couId not be moved. A Penrose drain was passed through the mesentery and the sigmoid coIon opened. A tube was inserted and hxed with a purse-string suture. CIosure of the abdomina1 waI1 was done in the usua1 manner. The operation occupied onehaIf hour. Prior to operation the patient was given 50,000 units of peniciIIin every three hours and streptomycin (2 gm. daiIy) in eight divided doses. This medication was continued. During the next week two unsuccessftd attempts were made on the ward to remove the gIass by recta1 manipuIation. MeanwhiIe the edema of the mucosa increased about the rim of the gIass and the area became very tender. Seven days after admission the patient was again operated upon under a spina anesthetic being given 150 mg. of procaine. A Iubricated vagina1 specuIum was inserted in the ana canal, the gIass exposed and a stee1 pIunger pIaced in the center of the gIass, which was then IiIIed with a quick-setting pIaster of paris and aIIowed to harden. When traction was appIied to this, the pIaster of paris moId came out but the gIass did not move. At this point a proctoIogic consuItation was obtained. A proctoscope was inserted into the rectum and thus into the gIass and the area inspected. There were some minor Iacerations and a Iarge edematous ring of mucosa, quite hemorrhagic, into which the remaining rim was embedded. The proctoscope was sIowIy withdrawn and the edematous ring ffattened with the edge of the scope. A snub-nosed Yeomans biopsy punch was carefuIIy inserted between the rim of the gIass and the edematous, somewhat ffattened mucosa1 ring, then pIaced under the thick rim of the gIass. By gentIe Ieverage aIong the rim of the gIass the vacuum was broken. The gIass became movabIe but further manipuIation and rotation was necessary to avoid any further Iaceration from the jagged broken edges. In order to avoid injury to the operator a CriIe retractor was shaped and pIaced in position between the gIass and the recta1 waI1 and the gIoved hand inserted behind the retractor. It was then possibIe to grasp the base of the gIass between the fingers. The gIass was rotated and carefuIIy extracted. A combination gauze pack was inserted against the previousIy infected Iacerations. The glass measured 8 cm. in Iength, 4 cm. in diameter at the bottom and 7 cm. in diameter at the open end. Two days Iater the rectum was again inspected and the ring of edematous mucosa found to be considerabIy reduced. The patient was referred to his surgeon and the coIostomy Iater cIosed.