Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.     

Lipopolysaccharide-induced cytokine production in peripheral blood mononuclear cells: intracellular localization of tumor necrosis factor α and interleukin 1β detected with a three-color immunofluorescence technique

 Lipopolysaccharide (LPS) can induce monocytes to produce various cytokines such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In the present study, the kinetics of both intracellular and extracellular accumulation of TNFα and IL-1β in LPS stimulated mononuclear cell (MNC) cultures has been determined. A three-color-immunofluorescence technique was used to detect intracellular accumulation of cytokines. Intracellular accumulation of TNFα in monocytes starts shortly after initiation of the culture; i.e., TNFα is detectable after 1 h, reaching a peak level after 3–4 hours with 50–65% of monocytes staining positive. In parallel with its increased intracellular presence, TNFα was also found in the culture supernatant. The intracellular accumulation of IL-1β in monocytes became detectable after 2 h of culture in the presence of LPS. After 4 h, a plateau was reached, with 90% of the monocytes being positive. In parallel, but with a little delay, IL-1β could be detected in the culture supernatant. TNFα and IL-1β can be produced simultaneously in the same monocytes as was shown by a three-color-immunofluorescence technique. It is concluded that TNFα and IL-1β are good parameters for the early measurement of monocyte activation and that both the intracellular accumulation in monocytes and the amount of secreted cytokines can be used for such a purpose. The intracellular accumulation in monocytes can be measured by the three-color-immunofluorescence technique described. Accepted: 27 August 1996     

Circadian and seasonal variation of the body temperature of sheep in a tropical environment

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55° C at 0500 hours in summer, and 38.87° C at 0600 hours in winter. The maximum was 40.03° C in summer (1700 hours) and 39.33° C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.     

PEST sequences in calmodulin-binding proteins

Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17kDa, acidic Ca²⁺-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences.     

Studies on the adenosine 3′,5′-monophosphate-dependent protein kinase of Blastocladiella emersonii

Using a combination of Chromatographic and sucrose density gradient techniques under carefully controlled conditions of pH and protease inhibitors, we demonstrate that there is only one form of adenosine 3′,5′-monophosphate-dependent protein kinase in the cytosol fraction of the Blastocladiella emersonii zoospore. If any of these conditions are omitted during extract preparation, one obtains what are apparently multiple forms of the enzyme, which are in reality artifacts due to extensive endogenous proteolytic activity. This endogenous protease is stimulated by alkaline pH and inhibited by antipain. The zoospore protein kinase is similar to type II protein kinase from mammalian cells in several aspects including Chromatographic behavior on DEAE-cellulose column, conditions for subunit dissociation and reassociation, as well as the molecular weight value of the regulatory subunit.     

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.     

Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS

The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N′,N′-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O₂-evolution and ¹⁴CO₂-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light-harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels.     

Differential effects of manganese ions on Blastocladiella emersonii adenylate cyclase.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity in Blastocladiella emersonii is associated with particulate subcellar fractions. Solubilization after treatment with detergent suggests its localization in a membrane fraction of the zoospore homogenate. The enzyme specifically requires Mn2+ for activity and is not stimulated by NaF. The kinetic characteristics of substrate utilization by B. emersonii adenylate cyclase were investigated with various concentrations of ATP and Mn2+, and in the presence of inhibitors. Plots of enzyme activity versus the actual concentration of the MnATP2- complex give sigmoid curves. An excess of Mn2+ activates the enzyme at low concentrations of substrate and leads to a modification of the enzyme kinetics. The nucleotides 5'-AMP and GTP were shown to be competitive inhibitors of the enzyme. In addition, kinetic data, obtained under conditions in which an inhibitor (ATP) is added in constant proportion to the variable substrate (MnATP2-) concentration, produced reciprocal plots that were linear and intersecting to the right of the ordinate, and secondary replots that were hyperbolic. These kinetic patterns support a model in which: MnATP2- is the substrate; free Mn2+ is an activator at low substrate concentrations, but an inhibitor at high substrate concentrations; and free ATP is not an efficient inhibiyor (Ki greater than 1.10(-4) M).