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.     

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)     

Separation of Bombyxin from a neuropeptide of Bombyx mori showing Summer-morph-producing Hormone (SMPH) activity in the Asian Comma Butterfly,Polygonia c-aureum L

A neuropeptide from brain-suboesophageal ganglion (Br-SG) complexes of the silkmoth, Bombyx mori, shows summer-morph-producing hormone (SMPH) activity in the Asian comma butterfly, P. c-aureum. The SMPH-active peptide was extracted and demonstrated to be almost the same molecular size as bombyxin (4-5kD), a nueropeptide which shows prothoracicotropic hormone (PTTH) activity when assayed in vitro with prothoracic glands (PGs) of 4th-instar B. mori larvae in vitro. A Sephadex G-50 fraction of 3-8kD molecules prepared from Br-SG complexes of B. mori adults was applied to CM-, SP-, DEAE- or QAE- Toyoperal columns at pH 5.6 (or pH 6.9). The SMPH-activity could be separated from the PTTH-activity (or bombyxin) by subjecting a SMPH- and PTTH-active preparation of B. mori to anion-exchange chromatography at pH 6.9. By reversed-phase HPLC following an anion-exchange chromatography, SMPH-activity was recovered in two fractions of 40-45% acetonitril. Results demonstrate that the B. mori peptide showing the SMPH-activity in P. c-aureum is a different molecule than bombyxin.     

Teratogenic effects of avidin-induced biotin deficiency in mice

Teratogenic effects of maternal biotin deficiency were examined at different levels of severity by adding three levels of avidin (10, 20, or 40 mg/kg) in the basal diet. There was a considerable increase of fetuses with multiple congenital malformations (micrognathia, cleft palate, and micromelia) with increasing amounts of avidin. The dose-response relationship was observable in the incidence of each malformation as well. The body weight of live fetuses was also significantly reduced. However, the dams did not exhibit any typical signs of biotin deficiency, such as loss of hair, dermatitis, or nervous irritability. These results suggest that biotin is important for early embryonic development in the mouse.     

Insuppressibility of plasma glucagon by orally or intravenously administered glucose in diabetes mellitus

In order to study the response of pancreatic alpha cells to the change blood glucose, plasma pancreatic glucagon levels were measured after glucose loading given orally (50g) or intravenously (25g) in twenty-two normal controls and eighty untreated diabetics. Basal plasma pancreatic glucagon levels did not differ significantly in the two groups. However, oral or intravenous glucose administration caused a decrease in plasma pancreatic glucagon in normal subjects but not in diabetics. In "moderate" or "severe" diabetics, plasma pancreatic glucagon tended to increase paradoxically following oral glucose loading. To evaluate the sensitivity of pancreatic alpha cells to glucose, we calculated the index, -sigma delta IRG/sigma delta BS, after oral glucose loading. It was 1.96 +/- 0.57 in normal subjects, and significantly higher than in "mild" (0.11 +/- 0.05), "moderate" (-0.002 +/- 0.06) and "severe" (-0.09 +/- 0.07) diabetics. These results demonstrate the insensitivity of alpha cells to hyperglycemia in patients with diabetes mellitus as compared with normal subjects.