p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.     

A Novel Macroinitiator for the Synthesis of Triblock Copolymers via Atom Transfer Radical Polymerization: Polystyrene‐block‐poly(bisphenol A carbonate)‐block‐polystyrene and Poly(methyl methacrylate)‐block‐poly(bisphenol A carbonate)‐block‐poly(methyl methacrylate)

Summary: Bis(hydroxy)telechelic bisphenol A polycarbonate (PC) was prepared via melt polycondensation of bisphenol A (BPA) and diphenyl carbonate (DPC) using lanthanum(III ) acetylacetonate as a catalyst for transesterification. Subsequently, the polycarbonate was converted to a bifunctional macroinitiator for atom transfer radical polymerization (ATRP) with the reagent, α‐chlorophenylacetyl chloride. The macroinitiator was used for the polymerization of styrene (S) and methyl methacrylate (MMA) to give PS‐block‐PC‐block‐PS and PMMA‐block‐PC‐block‐PMMA triblock copolymers. These block copolymers were characterized by NMR and GPC. When styrene and methyl methacrylate were used in large excess, significant shifts toward high molecular weights were observed with quantitative consumption of the macroinitiator. Several ligands were studied in combination with CuCl as the ATRP catalyst. Kinetic studies reveal the controlled nature of the polymerization reaction for all the ligands used.

Formation of a bifunctional ATRP macroinitiator by esterification of bis(hydroxy)telechelic PC with α‐chlorophenylacetyl chloride.     

Temperature dependence of M pilus formation as demonstrated by electron microscopy

The formation of IncM plasmid encoded pili is dependent on the incubation temperature of the corresponding host strains. By labelling the short, rigid M pili with the donor specific bacteriophage luminal diameter M, the presence of pili at 30 degrees C but not at 37 degrees C incubation temperature could be demonstrated for E. coli K12 substrains carrying different IncM group plasmids. In contrast, such a temperature dependence of M pilus formation is not observed in S. typhimurium substrains.     

Experimental pneumococcal meningitis: impaired clearance of bacteria from the blood due to increased apoptosis in the spleen in Bcl-2-deficient mice

Necrotic and apoptotic neuronal cell death can be found in pneumococcal meningitis. We investigated the role of Bcl-2 as an antiapoptotic gene product in pneumococcal meningitis using Bcl-2 knockout (Bcl-2(-/-)) mice. By using a model of pneumococcal meningitis induced by intracerebral infection, Bcl-2-deficient mice and control littermates were assessed by clinical score and a tight rope test at 0, 12, 24, 32, and 36 h after infection. Then mice were sacrificed, the bacterial titers in blood, spleen, and cerebellar homogenates were determined, and the brain and spleen were evaluated histologically. The Bcl-2-deficient mice developed more severe clinical illness, and there were significant differences in the clinical score at 24, 32, and 36 h and in the tight rope test at 12 and 32 h. The bacterial titers in the blood were greater in Bcl-2-deficient mice than in the controls (7.46 +/- 1.93 log CFU/ml versus 5.16 +/- 0.96 log CFU/ml [mean +/- standard deviation]; P < 0.01). Neuronal damage was most prominent in the hippocampal formation, but there were no significant differences between groups. In situ tailing revealed only a few apoptotic neurons in the brain. In the spleen, however, there were significantly more apoptotic leukocytes in Bcl-2-deficient mice than in controls (5,148 +/- 3,406 leukocytes/mm2 versus 1,070 +/- 395 leukocytes/mm2; P < 0.005). Bcl-2 appears to counteract sepsis-induced apoptosis of splenic lymphocytes, thereby enhancing clearance of bacteria from the blood.     

Dielectric properties of a combined main-chain/side-chain liquid-crystalline polymer

The dielectric relaxation properties of a combined main-chain/side-chain liquid-crystalline polymer were investigated. It was found that the rotation of the side chain about the main chain (δ-process) is not as strongly restricted as in side-chain liquid-crystalline polymers. This is attributed to the facts that the side chain is attached to the flexible spacer within the chain backbone and that the concentration of the side chains is comparatively small. Two low-temperature relaxation processes were observed to occur in the glassy smectic and the crystalline state. They are attributed to intramolecular motions with in the mesogenic groups.     

RPG1: an essential gene of Saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G₁ phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or α-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G₁ cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G₁ at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G₁ phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3. Received: 6 October 1997 / 8 November 1997     

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cold-adapted rats

The toxicity of 60g/kg 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD) given IP in corn oil/5% acetone was examined in male Sprague-Dawley rats adapted to 25 °C or 4 °C ambient temperature. Cold exposure significantly reduced mean time to death and tended to increase mortality. Body weight at the time of death was reduced at both ambient temperatures to about the same extent. Thus, the rate of body weight loss was about twice as fast in nonsurvivors at 4°C than at 25 °C. There was a continuous decrease in feed intake of the non-survivors at 25 °C until death. However, no reduction in feed intake occurred in any of the rats at 4 °C ambient temperature. At 14 days after dosing all TCDD-dosed animals were hypothyroid in terms of T₄ but essentially euthyroid in terms of T₃. Oxygen consumption at 10 days after dosing was reduced to the same extent in all TCDD-dosed rats without regard to survival status. By day 20 after TCDD dosage, survivors increased their oxygen consumption at both ambient temperatures to nearly control levels whereas non-survivors were unable to do so. Body temperature of all animals remained within normal range except for the non-survivors, which showed reduced rectal temperature shortly before death. It is concluded (1) that cold adaptation aggravates the toxicity of TCDD, (2) that reduced feed intake alone cannot explain TCDD-induced wasting syndrome, (3) that reduced oxygen consumption in TCDD-treated rats may be due to reduced feed intake and/or altered thyroid hormone status, and (4) that TCDD is likely to activate metabolic pathways which represent a wasteful utilization of ingested and/or stored energy.     

Investigations on neurotoxicity of chemical substances at the workplace

Summary A cross-sectional study was performed in order to investigate the influence of chronic lead-exposure on the peripheral nervous system. We examined 148 male workers of a storage battery manufacturing plant, who had been exposed to lead metal and inorganic lead compounds for 1 to 28 years (mean 11 years). Fifteen workers with non-occupational risks of peripheral neuropathy (former diseases, alcohol abuse, medication) were excluded from the study. The investigation program comprised: case history, physical examination, analyses of blood- and urine-samples and determination of maximal motor, mixed and sensory conduction velocity (NCV) of the ulnar and median nerve of the right forearm. Objectively no worker showed any signs of health effects related to lead exposure. The Biological Monitoring included the determination of (1) Blood-lead level (Pb-B), (2) Free erythrocyte porphyrins (FEP), (3) -Aminolevulinic acid dehydratase (ALA-D) and (4) -Aminolevulinic acid in urine (ALA-U). Further time-weighted-average (TWA)-values of Pb-B were calculated on the basis of several determinations over the period 1975–1981. The following actual (TWA) median values resulted: Pb-B 53 g/dl (54 g/dl), ALA-U 5.6 mg/l (8.4 mg/l), FEP 2.0 mg/l (2.0 mg/l). The Biologischer Arbeitsstoff Toleranz Wert (BAT) of 70 g//dl for Pb-B was exceeded in 15 workers (11%), and of 15 mg/l for ALA-U in 30 cases (23%). In comparison with age-matched controls, the lead workers showed a mild slowing of NCV with mean values between 0.8 and 2.0 m/s. Multiple stepwise regression analyses revealed statistically significant correlations between the four NCV and age as well as Pb-B. There were better correlations by using TWA than actual data of Pb-B. Consideration of the results of the regression analyses, together with an evaluation of the individual neurophysiological status as a function of internal lead exposure, a dose-effect-relationship was found only in the case of Pb-B exceeding 70 g/dl. From our study it is concluded that chronic lead exposure resulting in blood-lead levels of below 70 g/dl is no occupational risk causing a functionally significant slowing of nerve conduction velocities.With Grants from the Deutsche Forschungsgemeinschaft, Bonn (Project no. Va 23/19-1)