Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

A revision of current data and views on membrane hydrolysis and transport in the mammalian small intestine based on a comparison of techniques of chronic and acute experiments: experimental re-investigation and critical review

Literature data and the results of our investigations using both generally accepted and original perfusion techniques of the isolated loop of the rat small intestine in in vivo experiments are reviewed. Significant differences in the functioning of the small intestine under conditions of acute and chronic experiments are revealed. It has been established that in chronic experiments as compared to acute ones: (a) the absorption of glucose, galactose, fructose and glycine is 2-5 times higher; (b) Kt as well as Jmax values of the transport of these nutrients differ considerably; (c) Na+-independent mechanism of glucose and glycine transport predominates; (d) higher rates of membrane hydrolysis and more effective interactions between enzyme and transport systems of the enterocyte brush border membranes are observed; (e) functional characteristics of the small intestine affected by various experimental factors are more stable. The conclusion is made that it is necessary to revise current views of the scale and regularities of digestive-transport processes in the small intestine under physiological conditions. The importance of the suggested approaches for general and comparative physiology and biochemistry is discussed.     

Immunohistochemical detection of regulatory cells in the digestive system of leeches

The digestive system of four leech species,Glossiphonia complanata, Hirudo medicinalis, Haemopis sanguisuga, andErpobdella octoculata, was studied using, as markers, antisera to biologically active peptides, neurotensin, calcitonin, FRMF-amide, and serotonin. In the epithelium and the wall of the alimentary tract, regulatory cells were revealed. They differed in shapes and detected immunoreactivities; presumably they are elements of the diffuse endocrine system as well as of the peripheral nervous system. The data obtained show that the organization of the intestinal regulatory system in leeches basically is similar morphologically to intestinal regulatory systems of representatives of other classes of this type of invertebrate animals.     

Intestinal Regulatory Systems of Invertebrates and Their Probable Evolution in Multicellular Animals

A wide distribution of regulatory (endocrine, nerve) cells in the digestive tract epithelium has been shown in representatives of taxons at different levels of the evolutionary tree, and their brief comparative characteristics are presented. The hypothesis is put forward that the intestinal regulatory systems, on having initially appeared based on predominantly nerve elements, were subsequently complicated due to the appearance of various endocrine cells originating from the epithelial ones. In the course of evolution, the spatial separation occurred of this heterogeneous system into the intraepithelial endocrine and subepithelial nerve components, this process running in parallel in the rows of protostomian and deuterostomian animals.     

Chemiluminescent multiassay of pesticides with horseradish peroxidase as a label

Competitive chemiluminescent immunoassay based on a combination of five antibodies was used in a combination with neural network to identify and estimate amounts of three cross-reacting s-triazines (atrazine, terbythylazine and ametryn). Antibodies with different cross-reactivity towards s-triazines were immobilized in separate wells an eight-well microtiter strip. Training of neural networks was carried out with four different learning procedures. The best topology for the data measured was a net with two hidden layers with ten neurons in the first and 15 in the second layer trained with the Schmidhuber method. s-Triazine classification of environmental samples containing various analyte mixtures was correct in 70-100% of all cases depending on the type of analyte. The test developed can be proposed as an alternative field test for multianalyte environmental monitoring.     

Application of enzyme field-effect transistors for determination of glucose concentrations in blood serum

Glucose-sensitive enzyme field effect transistors (ENFETs) modified by an additional Nafion membrane have been developed and used for diluted blood samples analysis. The ENFET was used in the linear portion of the calibration curve up to 1.5 mM glucose in a model solution, which corresponds with up to 60 mM glucose in the undiluted samples (dilution 1:40). The high linearity of the Grans curve (factor of linearity is 1.03) obtained by the method of standard additions indicates the high precision of analysis. Glucose concentrations in different blood serum samples determined by ENFETs were compared with those measured by the commercial analyzer 'Eksan-G' and colorimetric method ('Diagluc' enzymatic kit), and good correlation between these methods was revealed. The high reproducibility and operational stability of the biosensor developed were demonstrated.     

Penetration of macromolecules into contracted blood clot.

The effectiveness of thrombolytic therapy is determined by accessibility of thrombus compartments to plasminogen activators and, therefore, depends on permeability of thrombus to blood born macromolecules. Accumulation of 125I labeled proteins with molecular massess ranging from 150 to 450 kD into partly contracted blood clot or plasma clot was consistent with diffusion coefficients 3.2 x 10(-11) and 2.7 x 10(-11) m2 s-1, respectively. So far as the model conditions imitated those for venous thrombi, these data indicate that such thrombi are porous enough for immunoconjugates of relatively big size.