Actin participates in the structure of liver intermediate filaments

A dominating protein fraction (p45) having molecular weight of 45000 and pI 5.45 was found in the intermediate filaments pellet obtained from rat liver besides the present cytokeratins. Peptide mapping and radioimmunological assays with antibodies against this protein and muscle actin proved that the p45 protein belongs to the actin group. Immunoelectron microscopy revealed that this protein is located on the liver intermediate filaments. By melting of the cytokeratin complexes in urea it was established that p45 protein is complexed with the low molecular weight cytokeratin.     

Synthesis of intrathecal interferon in systemic lupus erythematosus with neurological complications.

Intrathecal synthesis of interferon in the absence of viral or bacterial infection was detected during the occurrence of neurological complications in two patients with systemic lupus erythematosus. The interferons displayed characteristics similar to those observed in the sera of patients with the disease. No interferon inducing activity was detected in the cerebrospinal fluid or serum of the two patients. These observations support the hypothesis of a localised mechanism of interferon induction in systemic lupus erythematosus which includes the interaction of lymphocytes with damaged tissues.     

Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

Uteroferrin: A protein in search of a function

Uteroferrin, a purple-colored, iron-containing acid phosphatase, with many of the properties of a lysosomal hydrolase, transports iron from the mother to the conceptus in pregnant pigs. Uteroferrin, however, is but one member of what may be a broad class of iron-containing phosphatases with unusual spectral properties which result from a novel type of di-iron active site. The biological function of uteroferrin is unknown. We argue here that the in vivo function of uteroferrin, despite its undoubted ability to act as a potent acid phosphatase, is that of a transplacental iron transporter.     

Metabolism of carbon tetrachloride to phosgene

Aerobic incubation of hepatic microsomal fractions in the presence of carbon tetrachloride, NADPH and cysteine resulted in the formation of phosgene which was identified by gas chromatography/mass spectrometry as the adduct, 2-oxothiazolidine-4-carboxylic acid, formed by its reaction with cysteine. [¹³C]-Carbon tetrachloride was metabolized to 2-[¹³C]-oxothiazolidine-4-carboxylic acid the , when carbon tetrachloride was incubated in the presence of [¹⁸O]-O₂, 2- [¹⁸O]-oxothiazolidine-4-carboxylic acid was formed. The reaction was inhibited by carbon monoxide showing the involvement of the cytochrome P-450-dependent mixed function oxidase system. The metabolism of carbon tetrachloride to phosgene may play a role in the production of hepatotoxicity by this compound.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Novel inhibition of proteoglycan synthesis and exocytosis by diethylcarbamazine in the Swarm rat chondrocyte

Pretreatment of cultured chondrosarcoma chondrocytes at 37 degrees C for 15 min with 15 mM diethylcarbamazine (DEC) followed by a 60-min pulse with [35S] sulfate in the presence of DEC resulted in an approximate 40% inhibition of synthesis and a 75% inhibition of secretion of 35S-proteoglycan. The inhibition was dose-related and was not due to a decrease in protein synthesis. Chondrocytes exposed for 75 min to 15 mM DEC, washed, incubated for 17 h in DEC-free medium, and then pulsed with [35S]sulfate showed no inhibition in the rate of synthesis of proteoglycan or in the per cent of radiolabeled proteoglycans exocytosed into the culture medium, indicating full reversibility of the inhibitory effect. When chondrocytes were incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, secretion of beta-D-xyloside-bound 35S-glycosaminoglycan was inhibited by more than 70% despite an approximate 3-fold increase in intracellular 35S-macromolecules, as compared to cells exposed to beta-D-xyloside alone. Upon removal of DEC, the block in the secretion of beta-D-xyloside-bound 35S-glycosaminoglycans was reversed, although there was a 15-30-min lag in the initiation of exocytosis. Light and electron microscopic examination of chondrocytes after 75 min of incubation with 15 mM DEC revealed large vacuoles, a distended Golgi apparatus, and a distended endoplasmic reticulum which contained electron dense material. Upon removal of DEC, the vacuoles disappeared and distended organelles returned to their normal appearance between 15 and 30 min, coincident with the start of exocytosis of 35S-proteoglycan and beta-D-xyloside-bound 35S-glycosaminoglycan. These biochemical and morphological studies indicate that DEC treatment of chondrosarcoma chondrocytes alters the transport of molecules from the endoplasmic reticulum to the Golgi and the transport of molecules from the Golgi to the cell surface.