The diagnosis of antileptospiral antibodies in human subjects by solid-phase immunoenzyme analysis

The possibility of using the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of leptospirosis has been shown. This method has proved to be more simple and sensitive than the leptospiral microagglutination and lysis test. The data on obtaining genus-specific leptospiral antigens are presented. As revealed in this study, the antigens obtained by the complex treatment of microbial cells with ultrasound and detergents show the maximum activity in ELISA. The optimum parameters of the ELISA system for the diagnosis of leptospirosis have been established.     

Effects of death of a single neuron on the functional organization of the neuronal ensemble in the leech

The reaction of leech nervous system after elimination of individual mechanosensory neuron by intracellular Pronase injection were studied. In the motor neurons connected with killed cells at 14-90th days after the operation there were the changes of the resting membrane potential and amplitude of postsynaptic potentials developed by the stimulation of mechanosensory neuron. It is suggested that the leech nervous system serves as the dynamic formation depending on changes of neuronal ensemble structure.     

The possibility of using equine serum albumin in place of bovine serum albumin and ovalbumin in radioimmunological and immunoenzyme analyses and in virological practice

Horse serum albumin has been shown to meet the requirements to protein preparations for microanalysis and thus to be suitable for use in kits of reagents for the radioimmunological determination of insulin and myoglobin, for the determination of tick-borne encephalitis virus antigen by the method of the enzyme immunoassay and for the stabilization of proteins in the hemagglutination test and the hemagglutination inhibition test.     

The conformation of calmodulin: a substantial environmentally sensitive helical transition in Ca4-calmodulin with potential mechanistic function

The conformation of Ca4-calmodulin in solution, as assessed by far-UV peptide circular dichroism, contains significantly less alpha-helix than the proposed X-ray crystal structure. We now show that Ca4-calmodulin adopts significant additional helical structure in solution in the presence of a helicogenic solvent (50%, v/v, aqueous 2,2,2-trifluoroethanol or 50%, v/v, methylpentane-5,5-diol). We suggest that the long continuous helix (residues 66-92 of the crystal structure) is not necessarily a normal feature of the calmodulin structure in solution, and may be due in part to the conditions of crystallisation. This result is supported by time-resolved tyrosine fluorescence anisotropy studies indicating that Ca4-calmodulin in solution is an essentially compact globular structure which undergoes isotropic rotational motion. We conclude that, under appropriate ionic and apolar environmental conditions, Ca4-calmodulin undergoes a substantial helical transition, which may involve residues in the central region of the molecule. Such a transition could have an important function in determining specificity and affinity in interactions of calmodulin with different target sequences of Ca2+-dependent regulatory enzymes.     

The effects of p-mercuribenzenesulfonate on purified spectrin and actin

The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.     

Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.     

Polyproline binding is an essential function of human profilin in yeast.

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Delta. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.