Glutamine synthetase activity in the developing secondary palate and induction by dexamethasone

Glutamine synthetase (EC 6.3.1.2) (GS) and glutamyltransferase (EC 2.3.2.1) (GT) specific activity were examined in developing A/Jax and C57BL/6J (C57) mouse fetal secondary palates. In addition, the induction of palatal GS was also examined after maternal injection of dexamethasone. Palatal GT activity was uniformly higher in A/J than C57 palates with both strains showing highest activity late on day 13 of gestation and a drop in activity by early day 14. In contrast, A/J palatal GS activity peaked transiently late on day 13, dropped by early day 14 and remained lower throughout the remaining period of palatal development. Palatal GS activity in C57 mouse fetuses, although failing to show a discrete transient peak of activity, remained at a constant elevated level from early day 13 to late day 14 and did not decrease until day 15 of gestation. These elevated levels of palatal GS and GT activity correspond to the gestation period of maximal palatal glycoconjugate biosynthesis. Thus, palatal GS activity may play an important regulatory role in the synthesis of these macromolecules. A/J and C57BL/6J mice exhibit different susceptibilities to glucocorticoid-induced cleft palate. However, maternal administration of a non-teratogenic dose of dexamethasone on either late day 12 or late day 13 resulted in a dramatic stimulation of both A/J and C57 fetal palatal GS but not GT activity when assay 18 h later. A/J palatal tissue responded to dexamethasone with greater induction of palatal GS activity than enzyme activity in C57 palates. Palatal GS, sensitive to glucocorticoid stimulation, may thus be an important link in expressing hormonal control of normal palatal differentiation.     

Tissue Distribution,Gender- and Genotype-Dependent Expression of Autophagy-Related Genes in Avian Species

As a result of the genetic selection of broiler (meat-type breeders) chickens for enhanced growth rate and lower feed conversion ratio, it has become necessary to restrict feed intake. When broilers are fed ad libitum, they would become obese and suffer from several health-related problems. A vital adaptation to starvation is autophagy, a self-eating mechanism for recycling cellular constituents. The autophagy pathway has witnessed dramatic growth in the last few years and extensively studied in yeast and mammals however, there is a paucity of information in avian (non-mammalian) species. Here we characterized several genes involved in autophagosome initiation and elongation in Red Jungle fowl (Gallus gallus) and Japanese quail (coturnix coturnix Japonica). Both complexes are ubiquitously expressed in chicken and quail tissues (liver, leg and breast muscle, brain, gizzard, intestine, heart, lung, kidney, adipose tissue, ovary and testis). Alignment analysis showed high similarity (50.7 to 91.5%) between chicken autophagy-related genes and their mammalian orthologs. Phylogenetic analysis demonstrated that the evolutionary relationship between autophagy genes is consistent with the consensus view of vertebrate evolution. Interestingly, the expression of autophagy-related genes is tissue- and gender- dependent. Furthermore, using two experimental male quail lines divergently selected over 40 generations for low (resistant, R) or high (sensitive, S) stress response, we found that the expression of most studied genes are higher in R compared to S line. Together our results indicate that the autophagy pathway is a key molecular signature exhibited gender specific differences and likely plays an important role in response to stress in avian species.     

Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography.

Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.     

Characterization of soluble cyclic adenosine monophosphate-dependent protein kinase isozymes in murine embryonic palatal tissue

Certain hormonal primary messengers identified in the mammalian palate during its ontogeny transmit information to the interior of the cell via transmembrane signaling systems that control the production of the secondary messenger cyclic adenosine monophosphate. The singular role of intracellular cyclic AMP is to activate cAMP-dependent protein kinases (cAMP-dPK). cAMP-dPK were thus identified and characterized in the developing murine embryonic palate. Incubation of cytosolic fractions of embryonic palatal tissue with cAMP resulted in a dose-dependent increase in the cAMP-dPK activity ratio. A transient elevation of basal cAMP-dPK was seen during the period of palatal ontogeny that corresponded temporally with a previously demonstrated transient elevation of palatal basal cAMP levels. Fractions of embryonic palatal tissue cytosols derived by diethylaminoethyl (DEAE)-Sephacel chromatography were analyzed for phosphotransferase activity and for [3H]-cAMP binding to the regulatory (R) subunits of cAMP-dPK. Such analyses revealed two peaks of activity on day 13 of gestation. Based on the salt concentration at which the material in these peaks eluted from DEAE, its ability to cochromatograph with authentic cAMP-dPK isozymes, its molecular weight as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis, and the ability of the material to be photoaffinity labeled with [3H]-8-azidoadenosine 3',5' cyclic phosphate, types I and II cAMP-dPK were identified. Regulatory subunits of cAMP-dPK were characterized by the binding of [3H]-cAMP to cytosolic fractions of embryonic palatal tissue. Such binding was saturable (Bmax = 1,096 fmol/mg protein) and of high affinity (Kd = 7 nM). Only cAMP and cyclic guanosine monophosphate competed in a dose-related manner with [3H]-cAMP for binding to R subunits of cAMP-dPK. Adenosine, cTMP, and adenosine triphosphate, at doses up to 10(-4) M, did not compete for binding. Temporal analysis of binding data indicated that the number of binding sites transiently decreased during day 13 of gestation. Characterization of cAMP-dPK in tissue derived from the developing mammalian palate allows consideration of cAMP-dPK as a key regulatory enzyme capable of transducing hormonally elevated intracellular levels of cAMP into metabolic responses during orofacial ontogenesis.