Association of Brain Protein Phosphatase 1 with Cytoskeletal Targeting/Regulatory Subunits

Abstract: Protein phosphatase 1 catalytic subunit (PP1_C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)-labeled PP1_C revealed four major rat brain PP1_C-binding proteins (PP1bps) with molecular masses of ≈216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1_C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size-exclusion chromatography of solubilized extracts separated two main PP1 activities (≈600 and ≈100 kDa). PP1bps and PP1_Cγ₁ were enriched in the ≈600-kDa peak, but PP1_Cβ was enriched in the ≈100-kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG-PP1_Cβ than recombinant DIG-PP1_Cγ₁ or DIG-PP1_Cα. Solubilized PP1bp175 and PP1bp134 interact with PP1_C under native conditions, because they both (1) coeluted from size-exclusion and ion-exchange columns; (2) bound to microcystin-LR-Sepharose; and (3) coprecipitated using PP1_C antibodies. Trypsinolysis of the ≈600-kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1_C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform-selective binding of PP1_C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.     

Synthesis and properties of a new cleavable nucleic acid-protein crosslinking reagent

A new compound, dithiobis[9-(2-ethylenecarbamoylethylamino)-2,3-dimethoxy-6-azido-acridine], was synthesized and used in some preliminary experiments to form cleavable complexes between nucleic acids and proteins. In a first step the azidoacridine moiety of the reagent intercalates between the bases of nucleic acids and is then bound by reaction of the azido group. The disulfide group of the reagent is simultaneously converted under reducing conditions into a thiol which, in a second step, can be bound by oxidation to -SH groups of a vicinal protein (additional -SH groups can be inserted in the protein using 2-iminothiolane). The nucleic acid-protein complexes thus formed can be redissociated by reduction. The potential applications of this new cleavable crosslinking reagent could be extended to topographical investigations of any biological structure composed of nucleic acids and proteins.     

Transforming growth factor beta 2 differentially modulates interleukin-1 beta- and tumour-necrosis-factor-alpha-stimulated phospholipase A2 and prostaglandin E2 synthesis in rat renal mesangial cells.

Isolated hepatocytes from streptozotocin-diabetic rats failed to respond to a glucose load with an activation of glycogen synthase. This lesion was associated with severely decreased activities of glycogen-synthase phosphatase and of glucokinase. All these defects were abolished after consumption for 13-18 days of drinking water containing Na3VO4 (0.7 mg/ml), and they were partially restored after 3.5 days, when the blood glucose concentration was already normalized. In all conditions the maximal extent of activation of glycogen synthase in cells closely parallelled the activity of glycogen-synthase phosphatase.     

Revised location of the Escherichia coli gene coding for ribosomal proteins S2

Summary P1 transduction studies of a mutation of Escherichia coli affecting the rpsB gene previously suggested the map order popC-rpsB-dapD. Biochemical analysis of transductants has now revealed that the rpsB gene lies in fact to the right hand (tsf) side of the dapD marker.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

The influence of trace elements on the composting of wheat straw

Бор и молибдеп в качестве микроэлементов, а также серпокислый аммоний и азотнокислый натрий в качсетве источников азота оказывали влияние на скорость компостирования пшеничной соломы. Источники азота применялись в таком количестве, что к соломе был прибавлен 1% азота. В опытах с азотнокислым натрием оптимальпая концентрация молибдена составляла 5 р. p. m., с сернокислым аммонием – 1 р. p. m. Максимальное уменьшение количества сухого вещества через 60 дней составляло 50% и было отмечено с азотнокслым натриемпосле прибавления 1 p. p. m. Бора. За пределами оптимального уровня наблюдалось угнетающее или токсическое действие прибавления микроэлемента. Вопрос, целсообразно ли нбавление бора имолибдсна для ускорения компостирования, зависит от их концентрации в органических остатках, в источниках азота, в инокулюоме и в применяемой воде. Значение микроэлементов в энзиматических системах бактерий хорошо известно, но для определения их требований к микроэлементам необходимы более детальные опыты. Несмотря на общераспростаненый взгляд, что растительный материал содеожит достаточное количество микрозлементов для оьеспечения его разложния, предлагаемая работа говорит о том, что для быстрого кобавлять известные их количества, так как во всех применявшихся концентрациях бор и молибден вызывали более значтельныс потерн сухого вещества и общего углерода иконсрвирование более значительного количества общего азота, чем в контроле.