Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain

Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

Regulation of substrate oxidation in isolated myocardial cells by beta-hydroxybutyrate

The role of ketone bodies in myocardial substrate oxidation was examined using freshly isolated Ca2+-tolerant heart myocytes, beta-hydroxybutyrate (beta OHB) inhibited lactate oxidation by the myocytes by 30-60%, and the inhibition was concentration dependent. Palmitate oxidation was also markedly decreased, whereas octanoate oxidation was only minimally affected by the presence of beta OHB. Lactate, octanoate, or palmitate had little, if any, effect on beta OHB oxidation. beta OHB oxidation was reduced by 22-28% in myocytes isolated from chronically diabetic rats, whereas the oxidation of palmitate remained similar to the controls. However, beta OHB still inhibited palmitate oxidation to the same extent as in the control cells. Our data support the role of beta OHB as a physiologic regulator of myocardial substrate metabolism.     

Synchronization of estrus after treatment with Luprostiol in beef cows and in beef and dairy heifers

Four trials were conducted to study synchronous estrous response in beef cows and in beef and dairy heifers to Luprostiol (13, thia-PG-F(2)alpha analog) in comparison with other prostaglandin products. In Trial 1, 60 virgin beef heifers were observed for estrus for 5 d and artificially inseminated. Heifers not observed in estrus within 5 d were randomly assigned to receive 15 mg Luprostiol or 25 mg Lutalyse. In Trial 2, 75 multiparous, lactating beef cows were randomly assigned to receive either 15 mg Luprostiol, 25 mg Lutalyse or 500 mcg Estrumate. All cows received a second injection of the respective treatment 11 d later. In Trial 3, 96 multiparous, lactating beef cows were randomly assigned to receive 15 mg Luprostiol or 25 mg Lutalyse. All cows received a second injection of the respective treatment 11 d later. In Trial 4, virgin dairy heifers were palpated per rectum. Seventy-seven heifers with a palpable corpus luteum (CL) were randomly assigned to receive 15 mg Luprostiol or 500 mcg Estrumate. In all trials animals were artificially inseminated 12 h following observed estrus. Estrous response during the 5-d synchronized period was 44% for Luprostiol and 42% for Lutalyse treated heifers in Trial 1. It was 52, 56 and 60%, respectively, for Luprostiol, Lutalyse and Estrumate treated cows in Trial 2; 23% for Luprostiol and 19% for Lutalyse treated cows in Trial 3; and 68% for Luprostiol and 70% for Estrumate treated heifers in Trial 4. Treatment with Luprostiol results in a similar synchronous estrous response as with the other prostaglandin products used in these studies.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.     

Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Effect of dietary lipid supplementation on progesterone concentration and reproductive performance in suckled beef cows

Using whole cottonseed as a lipid source, silage-based diets that were isocaloric and isonitrogenous yet varied in lipid level were fed to multiparous cows. In Experiment 1, 48 cows (n = 12 per treatment) were allotted to 1 of 4 treatments where diets were formulated to supply 3.9, 4.3, 5.3 and 6.3% of total lipid. In Experiment 2, 66 cows (n = 22 per treatment) were allotted to 1 of 3 treatments where diets were formulated to supply 3.1, 5.5 and 8.3% of total lipid. Length of the first ovarian cycle, length of the first normal estrous cycle, postpartum intervals to onset of ovarian luteal activity and to first estrus were not affected by diet (P>0.10) in either experiment. Mean progesterone (P(4)) concentrations for first normal estrous cycles were not different (P>0.10) in either experiment. Anestrous periods were divided into 3 phases for analyses: Phase I) parturition to onset of ovarian luteal activity, Phase II) first ovarian luteal activity and Phase III) first normal estrous cycle. No differences were observed in P(4) concentrations during any phase of the postpartum period. In conclusion, isocaloric and isonitrogenous diets with increasing levels of lipid had no effect on reproductive performance in suckled beef in these experiments.     

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations A_o binding site concentration - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-nor-8-hydroxyfusicoccin - K_D dissociation constant of the FCBP-radioligand complex We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision).