ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.     

Relationships of food uptake and body components of Calanus finmarchicus,C. glacialis and C. hyperboreus to particulate matter and water characteristics in Fram Strait

Summary During MIZEX'83 and MIZEX'84 food composition and food uptake by three Calanus species were investigated in the northern Greenland Sea. Samples were obtained from open water, the marginal ice zone, the pack ice region and the East Greenland shelf polynya. Food uptake of Calanus spp. was determined in ship-board incubation experiments under in situ conditions and volume of particulate matter in the upper 300 m of the water column was measured. Principal components analysis was used for data evaluation. Under the pack ice strongly reduced amounts of particulate matter in the euphotic zone were found while concentrations were elevated in the marginal ice zone and the East Greenland shelf polynya. Food uptake of C. finmarchicus was correlated to food concentration. In the pack ice region ingestion was close to zero for all species investigated. Likewise body weights were significantly lower than in MIZ or polynya. Principal components analysis of physical data shows a clustering of MIZ station groups at high temperature and salinity, clearly separated from pack ice and polynya stations. Analysis of biological data results in the same grouping of stations demonstrating a strong influence of hydrographic conditions on plankton development. It is concluded that herbivorous copepods cannot sustain themselves in the pack ice region but only at places of high productivity like marginal ice zone and East Greenland shelf polynya.     

Altered drug translocation mediated by the MDR protein: Direct,indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ltpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential () and/or intracellular pH (pH_i) and therebyindirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pH_i, or -dependent manner. A third model proposes that the protein alternates between drug pump and Cl^– channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pH_i, , and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.     

Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex

The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9–15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35±13 OAP/m², corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22±12 OAP/², corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another mode of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130±136 IMP/ m²) than in membranes of astrocytes in the center of the slice (700±172 IMP/m²). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.     

Rapid purification of DesPro(2)-Val15-Leu17-aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae

A rapid two-step procedure has been developed for the purification of Despro(2)-Val15-Leu17-aprotinin from the culture supernatant of a recombinant yeast by affinity and ion-exchange chromatography. DesPro(2)-Val15-Leu17-aprotinin was purified to homogeneity, as demonstrated by dodecylsulfate gel electrophoresis and analysis of the N-terminal amino acid sequence. (c) 1993 John Wiley & Sons, Inc.     

Dynamic properties of cortical evoked (10 Hz) oscillations: theory and experiment

Experiments probed the dynamic properties of stimulus-evoked (10 Hz) oscillations in somatosensory cortex of anesthetized rats. Experimental paradigms and statistical time series analysis were based on theoretical ideas from a dynamic approach to temporal patterns of neuronal activity. From the results of a double-stimulus paradigm we conclude that the neuronal response contains two components with different dynamics and different coupling to the stimulus. Based on this result a quantitative dynamic model is derived, making use of normal form theory for bifurcating vector fields. The variables used are abstract, but measurable, dynamic components. The model parameters capture the dynamic properties of neuronal response and are related to experimental results. A structural interpretation of the model can be given in terms of the collective dynamics of neuronal groups, their mutual interaction, and their coupling to peripheral stimuli. The model predicts the stimulusdependent lifetime of the oscillations as observed in experiment. We show that this prediction relies on the basic concept of dynamic bistability and does not depend on the modeling details.