Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 microm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 microm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 microm, but not 1 microm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50-100% and PKB activity 2-4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.     

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein,survival of motor neuron

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.     

Genomic structures and population histories of linguistically distinct tribal groups of India

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.     

Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.     

Mitochondrial calcium in relaxed and tetanized myocardium.

The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+].     

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2

Gao, Yuansheng, Jean-François Tolsa, Hai Shen, and J. Usha Raj. Effect of selective phosphodiesteraseinhibitors on response of ovine pulmonary arteries to prostaglandinE₂. J. Appl. Physiol. 84(1): 13-18, 1998.Several adenosine3,5-cyclic monophosphate (cAMP)-hydrolyzingphosphodiesterase isozymes are present in the pulmonary vasculature.The present study was designed to determine the effect of selectiveinhibitors of phosphodiesterase subtypes on prostaglandinE₂(PGE₂)-induced relaxation ofisolated fourth- generation pulmonary arteries of newborn lambs.PGE₂ and forskolin causedpulmonary arteries to relax and induced an increase in theintracellular cAMP content in the vessels. The relaxation and change incAMP content were augmented by milrinone and rolipram, inhibitors ofphosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. Theaugmentation in relaxation and the increase in cAMP content caused bymilrinone plus rolipram was greater than the sum of theresponses caused by either of the inhibitors alone.8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation andchange in cAMP induced by PGE₂ andforskolin. Acetylcholine alone had no effect on cAMP content in thevessels but augmented the relaxation and the increase in cAMP inducedby PGE₂ and forskolin in arterieswith endothelium. This effect was not observed in arteries withoutendothelium or in arteries with endothelium treated withN^G-nitro-L-arginine.These results suggest that PDE3 and PDE4 are the primary enzymeshydrolyzing cAMP of pulmonary arteries of newborn lambs and that aninhibition of both PDE3 and PDE4 would result in a greater effect thanthat caused by inhibition of either one of the subtype isozymes alone.Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediatedrelaxation by inhibition of PDE3.