Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.     

Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination.

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.     

Agglutination, Adherence, and Root Colonization by Fluorescent Pseudomonads

Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10⁶ Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10⁵ to 10⁶ Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg^- mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.     

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).     

Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725

Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO₂). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity.     

Polarized fluorescence and absorption of macroscopically aligned Light Harvesting Complex II.

Polarized absorption and fluorescence measurements have been performed at 77 K on isotropic and anisotropic preparations of trimeric Light Harvesting Complex II (LHC-II) from spinach. The results enable a decomposition of the absorption spectrum into components parallel and perpendicular to the trimeric plane. For the first time, it is shown quantitatively that the strong absorption band around 676 nm is polarized essentially parallel to the plane of the trimer, i.e., the average angle between the corresponding transition dipole moments and this plane is at most 12 degrees. The different absorption bands for LHC-II should not be considered as corresponding to individual pigments but to collective excitations of different pigments. Nevertheless, the average angle between the Qy transition dipole moments of all chlorophyll a pigments in LHC-II and the trimeric plane could be determined and was found to be 17.5 degrees +/- 2.5 degrees. For the chlorophyll b pigments, this angle is significantly larger (close to 35 degrees). At 77 K, most of the fluorescence stems from a weak band above 676 nm and the corresponding transition dipole moments are oriented further out of plane than the dipole moments corresponding to the 676-nm band. The results are shown to be of crucial significance for understanding the relation between the LHC-II structure and its spectroscopy.     

The consequences of a drastic fish stock reduction in the large and shallow Lake Wolderwijd,The Netherlands. Can we understand what happened?

In 1990 an experiment started in the large and shallow lake Wolderwijd (2700 ha, mean depth 1.5 m) to improve the water quality. About 75% of the fish stock was removed (425 000 kg fish). The fish was mainly composed of bream and roach. In May 600000 young pikes (3–4 cm) were introduced.In May 1991 the water became very clear (Secchi depth 1.8 m) during a spring bloom of large Daphnia. Then the grazing by zooplankton was eight times higher than the primary production of algae and the total suspended matter concentration became very low. Compared to the situation before the fish reduction, the grazing had increased only slightly, while the primary production had decreased significantly in early spring. The fish stock reduction might have contributed to the reduction in primary production by a reduced internal nutrient load. The clear water period lasted six weeks. Daphnia disappeared in July due to food limitation, the algal biomass increased and the Secchi depth became 50 cm. Daphnia did not recover during summer, due to predation that was not caused by 0 + fish but by the mysid shrimp Neomysis integer. Neomysis could develop abundantly, because of the reduced biomass of the predator perch. The production of young fish had been low because of the cold spring weather. The cold weather was probably also responsible for the slow increase in density of macrophytes. After 1991, perch probably can control Neomysis. Due to lack of spawning places and shelter for 0 + pike, pike was probably not able to control the production of 0 + fish. In a lake of this scale, it will not be easy to get more than 50% coverage of macrophytes, which seems necessary to keep the algal biomass low by nutrient competition. Therefore, we expect also in the future a decrease in transparency in the summer. Locally, especially near Characeae, the water might stay clear.