Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data

Journal of Plant Research - A group of temperate grassland plant species termed the “Mansen elements” occurs in Japan and is widely distributed in the grasslands of continental East...     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

Repeated batch production of alkaline protease by solid-state fermentation using urethane foam as carriers

By immobilizing fungi on a urethane foam carrier (UFC), a novel solid-state fermentation system was developed in order to produce repeatedly industrial useful enzymes. In this study, alkaline protease was produced by growing Aspergillus oryzae 460 (ATCC 20386) in a flask scale. Repeated batch production of alkaline protease was carried out by exchanging a part of the culture broth with fresh medium every 12 hr. The effects of feeding medium composition, and feeding volume were examined. Alkaline protease production stopped in the early phase at high values of soluble starch feeding rate and culture broth dilution rate. The enzyme production continued longer when 10 to 30 g/l polypepton was added to the feeding medium. Using soluble starch solution as feeding medium, a maximum activity of 520,000 U/l-bulk volume alkaline protease was obtained at culture time of 168 hr where the culture conditions of soluble starch concentration and feeding volume were 100 g/l and 0.025 l/l-bulk volume/dose, respectively. Production of the enzyme continued for 300 hr and total aklaline protease activity reached 870,000 U/l-bulk volume by adding 30 g/l polypepton to the fresh medium.     

Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery.

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.     

Suppression of Quantum Yield of Photosystem II by Hyperosmotic Stress in Chlamydomonas reinhardtii

Addition of ethylene glycol (EG) or NaCl to cells of Chlamydomonasreinhardtii induced transient non-photochemical quenching ofChl fluorescence correlated with the inhibition of photosyntheticoxygen evolution. The induction of the quenching and subsequentrecovery proceeded not only in the light but also in the dark.The quenching was almost unaffected by the protonophore nigericin,suggesting the involvement of a type of non-photochemical quenchingattributable to a state 2 transition. Higher concentrationsof EG or NaCl caused a delay of the recovery of the maximumfluorescence yield (Fm'). Dark reduction rate of P700⁺ afterthe application of a flash light in the presence of DCMU wasenhanced by the hyperosmotic shock, suggesting a stimulatedreduction of the intersystem electron carriers. It is proposedthat the osmotic stress stimulates electron donation from stromalcomponents via the NAD(P)H dehydrogenase, which results in thereduction of the intersystem chain and triggering of a state2 transition leading to stimulated cyclic PSI activity. (Received May 16, 1995; Accepted July 26, 1995)     

Possible involvement of ethylene-activated {beta}-cyanoalanine synthase in the regulation of cocklebur seed germination

A possible involvement of ß-cyanoalanine synthase(CAS: EC 4.4.1.9 [EC] ) in germination processes of seeds was demonstratedusing pre-soaked upper seeds of cocklebur (Xanthium pennsylvanicumWallr.). Pretreatment in anoxia not only with KCN but also cysteine,as the substrates for CAS, stimulated the subsequent germinationof cocklebur seeds in air. However, the effect of cysteine wasmanifested even in air when applied together with C₂H₄, andits effect was further enhanced in combination with KCN. Thegermination-stimulating effect of KCN was intensified by C₂H₄only when 0₂ was present. In contrast, serine, another substrateof CAS, was effective in air only when combined with C₂H₄ and/orKCN. The addition of cysteine greatly reduced the cyanogenicglycoside content of seeds, but increased HCN evolution. Onthe other hand, glutathione did not have any effect on cockleburseed germination, HCN evolution or bound cyanogen content, suggestingthat cysteine is not acting as a reducing reagent. It is suggestedthat CAS regulates the process of cocklebur seed germinationby the dual action of enlarging the pool of amino acids andsupplying sulphydryl bases, the latter being more determinatelyimportant. Serine is effective only via the former action, whilecysteine would act via both. Key words: Cyanide, cyanogenic glycoside, ß-cyanoalanine synthase, seed germination, Xanthium pennsylvanicum