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Peptides as modifiers of Na+-induced pinocytosis in starved Amoeba proteus.

Low concentrations of six peptide hormones; glucagon, vasoactive intestinal peptide, substance P, angiotensin II, lysine-vasopressin, arginine-vasopressin, and the chemotactic peptide fMet-Leu-Phe, activated the capacity for pinocytosis in starved Amoeba proteus. Competitive inhibitors of the chemotactic peptide in leucocytes inhibited activation by fMet-Leu-Phe, suggesting that its action in the amoeba is mediated by specific receptors. The opioid peptides, beta-endorphin, dynorphin (1-13) and leu-enkephalin abolished through a naloxone-sensitive mechanism activation by hormones and several other activating agents. Also, low concentrations of beef and pork insulin inhibited activation by peptide hormones. An insulin analogue of low potency in mammalian cells was inactive in the amoeba. These results support the hypothesis that besides opioid receptors, there may be insulin receptors and possibly receptors for several other peptide hormones in Amoeba proteus.

Amoeba↗

Photophobic and phototactic responses of Amoeba proteus in KCN and SHAM solutions.

The effects of the metabolic inhibitors KCN and SHAM on phototaxis and photophobic response in Amoeba proteus have been studied. Both drugs neither change amoebae photophobic response nor the phototactic reaction. The results indicate clearly that the negative phototactic orientation is not impaired by impediment of respiration thus it is not directly coupled to the differences in energy production in different parts of A. proteus body.

Amoeba↗

Structure and evolution of the mitochondrial control region and flanking sequences in the European cave salamander Proteus anguinus.

The European cave salamander Proteus anguinus Laurenti 1768 is one of the best-known subterranean animals, yet its evolutionary history and systematic relationships remain enigmatic. This is the first comprehensive study on molecular evolution within the taxon, using an mtDNA segment containing the control region (CR) and adjacent sequences. Two to seven tandem repeats of 24-32 bp were found in the intergenic spacer region (VNTR1), and three, four or six repeats, 59-77 bp each, in the 3' end of the CR (VNTR2). Different molecular mechanisms account for VNTR2 formation in different lineages of Proteus. The overall CR variation was lower than that of the spacer region, the 3' end of the cytb gene, or the tRNA genes. Individual genes and the concatenated non-repetitive sequences produced similar, well resolved maximum likelihood, Bayesian inference and parsimony trees. The numbers of repeat elements as well as the genealogy of the VNTR2 repeat units were mostly inconsistent with the groupings of the non-repetitive sequences. Different degrees of repeat array homogenization were detected in all major groups. Orthology was established for the first and the second VNTR2 elements of some populations. These two copies may therefore be used for analyses at the population level. The pattern of CR sequence variation points to strong genetic isolation of hydrographically separated populations. Genetic separation of the major groups of populations is incongruent with the current division into subspecies.

Animals↗

Calcium distribution in Amoeba proteus.

A preliminary investigation of the distribution of cellular calcium in Amoeba proteus was undertaken. Total cellular calcium under control conditions was found to be 4.59 mmol/kg of cells. When the external Ca++ concentration is increased from the control level of 0.03 to 20 mM, a net Ca++ influx results with a new steady-state cellular calcium level being achieved in integral of 3 h. At steady state the amount of calcium per unit weight of cells is higher than the amount of calcium per unit weight of external solution when the external concentration of Ca++ is below 10 mM. At external Ca++ concentrations above this level, total cellular calcium approaches the medium level of Ca++. Steady-state calcium exchange in Amoeba proteus was determined with 45Ca. There is an immediate and rapid exchange of integral of 0.84 mmol/kg of cells or 18% of the total cellular calcium with the labelled Ca++. Following this initial exchange, there was very little if any further exchange observed. Most of this exchanged calcium could be eliminated from the cell with 1 mM La+++, suggesting that the exchanged calcium is associated with the surface of the cell. Increase in either the external Ca++ concentration of pH raise the amount of exchangeable calcium associated with the cell. Calcium may be associated with the cell surface as a co-ion in the diffuse double layer or bound to fixed negative sites on the surface of the cell. If Ca++-binding sites do exist on the cell surface, there may be more than one type and they may have different dissociation constants. The cytoplasmic Ca++ ion activity is probably maintained at very low levels.

Amoeba↗

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium.

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.

Amoeba↗

PROTEUS: a suite of programs for prediction of structural features of proteins using an Apple IIe.

We have implemented several algorithms, developed by various authors for predicting structural features of proteins from their primary structure, on an Apple IIe and collected them in a suite, named PROTEUS. This suite incorporates: (i) methods for predicting secondary structure; (ii) the algorithm for computing the hydropathy profile using one out of five available sets of parameters; (iii) the algorithms for calculating the hydrophobic moment plot; and (iv) for performing the amphipathic analysis using one out of four available sets of parameters. The suite has a utility program for storing on a disk the sequence to be analysed. As an example, we applied some of the methods included in PROTEUS to predict the structure of a mitochondrial leader peptide. The results suggest the occurrence of structural features possibly related to the import of proteins into mitochondria.

Algorithms↗

Phylogenetic characterization of Legionella-like endosymbiotic X-bacteria in Amoeba proteus: a proposal for 'Candidatus Legionella jeonii' sp. nov.

The X-bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro, the identity of X-bacteria has not been determined. As the life cycle of X-bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA-targeted probe. In addition we cloned RNA polymerase beta-subunit gene (rpoB) of X-bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella. Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X-bacteria to be a Legionella sp. In all three phylogenic analyses, X-bacterium was placed apart from Legionella-like amoebal pathogens present in soil amoebae. Thus, we propose the name 'Candidatus Legionella jeonii' sp. nov. for the endosymbiotic X-bacteria in Amoeba proteus.

Amino Acid Sequence↗

Characterization of sams genes of Amoeba proteus and the endosymbiotic X-bacteria.

As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S-adenosylmethionine synthetase (SAMS). When symbiont-free D amoebae are infected with symbionts (X-bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long-established xD amoebae contain an intact sams gene and thus the loss of xD amoeba's SAMS is not due to the loss of the gene itself. The open reading frame of the amoeba's sams gene has 1,281 nucleotides, encoding SAMS of 426 amino acids with a mass of 48 kDa and pI of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5'-flanking region of amoeba sams contains consensus-binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbiont's sams gene is also presented. The open reading frame of X-bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pI of 6.0. The X-bacteria SAMS has 45% sequence identity with that of A. proteus.

Amino Acid Sequence↗

Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.

Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.

Amino Acid Sequence↗

Biochemical and Molecular Characterization of Obesumbacterium proteus, a Common Contaminant of Brewing Yeasts.

We have evaluated the effectiveness of API 20E, Biolog testing, plasmid profiling, ribotyping, and enteric repetitive intergenic consensus (ERIC)-PCR to characterize, classify, and differentiate nine bacterial isolates of the common brewery contaminant Obesumbacterium proteus. Of the five typing techniques, Biolog testing, plasmid profiling, and ERIC-PCR provided the most differentiation, and API 20E testing and ribotyping were relatively indiscriminate. The molecular biology approach of ERIC-PCR offered the ideal combination of speed, simplicity, and discrimination in this study. Overall, the results are supportive of the view that O. proteus can be subdivided into two biogroups, biogroup 1, which has considerable biochemical and genetic homology to Hafnia alvei, and biogroup 2, which is relatively heterogeneous.

Journal Article↗

Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, beta-Phenethyl Alcohol, and p-Nitrophenylglycerine.

The effects of sodium tetradecyl sulfate (STS), beta-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.

Journal Article↗

Permanent alterations of the L-forms of proteus and salmonella under various conditions.

L-forms obtained from three strains of Proteus and from one strain of Salmonella have been kept for 15 to 20 years by weekly or monthly transfers on agar plates containing penicillin. The morphology and growth requirements of these strains have changed. They now grow abundantly on the surface of agar and in broth. The cultures consist of large bodies, small granules, and transitional forms. These organisms are more resistant to distortion and stain more deeply than organisms of the usual L-forms. In broth and to a lesser extent on agar, branching filaments develop, on the ends of which both the large, round organisms and small organisms are produced. The filaments are a transitional stage in the development of the cultures. Usual bacillary forms were not present in the culture and did not appear in successive transfers in the absence of penicillin. Bacilli reappeared on exposure of the L cultures to the influence of a spore-bearing bacillus. A similar transformation of L-forms has also been observed developing within a short time in recently isolated A and B type L cultures of one Proteus strain during the process of reversion to the bacterial form. The altered cultures are fixed in a stage of transition between the B type L-form and the regular bacteria.

Journal Article↗

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus.

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca2+ in the external medium. Increasing external Ca2+ to approximately 10(-4) M increases pinocytotic intensity, while increases in Ca2+ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca2+ at the surface of Amoeba proteus was therefore examined. Alcian blue and Na+, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca2+ in the range of 3 X 10(-5) to 1.5 X 10(-4) M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca2+ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity.

Alcian Blue↗

Behavioral sensitivity of the European blind cave salamander, Proteus anguinus, and a Pyrenean newt, Euproctus asper, to electrical fields in water.

In the urodeles Proteus anguinus and Euproctus asper, thresholds of an overt avoidance response to weak electrical field stimuli (continuous sine-waves) were measured as a function of frequency. Thresholds down to 0.1 mV/cm (30 nA/cm2) were found in P. anguinus and 2 mV/cm (600 nA/cm2) in E. asper at 'best frequencies' (B.F.) of 20-30 Hz, but sensitivity covered a total frequency range of below 0.1 Hz to 1-2 kHz, with up to 70 dB higher thresholds. Average thresholds of 1 mV/cm in P. anguinus and 40 mV/cm in E. asper were more than 30 dB apart and significantly different. Both species were sensitive to galvanic DC-pulses, clicks, and noise bursts with intensities of about the same order of magnitude. Specimens of the transparent catfish, Kryptopterus (Siluridae) reacted in the same frequency range as found for Proteus and Euproctus, and had still lower thresholds, down to 0.02 mV/cm (1.5 nA/cm2). The biological significance and possibly still ongoing evolution of the electrical sense in urodeles is interpreted in terms of comparative sensory physiology and more recent, still speculative, evolutionary diversification during and since the Pleistocene.

Animals↗

Behavioral evidence and supporting electrophysiological observations for electroreception in the blind cave salamander, Proteus anguinus (Urodela).

Conditioning experiments revealed that Proteus perceives a back-and-forth moving (approximately 1 Hz) direct-current field and its polarity. Minimum behavioral thresholds occurred at a current density of 0.15 microA/cm2, corresponding to a voltage gradient of 0.5 mV/cm. Recordings from afferent nerve fibers showed that ampullary electroreceptors in Proteus respond as do other nonteleost receptors, i.e. with an increase in discharge rate to cathodal current and a decrease to anodal current (threshold: approximately 1 mV/cm).

Afferent Pathways↗

Persistence of retinal dopamine cells in the degenerated eye of the cave salamander, Proteus anguinus L.

The Proteus anguinus L. is a blind cave perennibranch amphibian whose visual system undergoes an important morphogenetic degeneration in adulthood. The eyeball becomes atrophied and disappears under the fat tissue of the head. However, a retina can still be identified and a photophobic behavior of the animal indicates a remaining photosensitivity. In the oldest animal observed, some photoreceptor cells are still present as well as other types of retinal neurons. Characteristic synapses are observed in both the inner and outer plexiform layers. Dopaminergic amacrine cells, with processes in the inner plexiform layer, can be identified by their tyrosine-hydroxylase immunoreactivity. Taken together, these results indicate a possible functional role of the remaining retina. Since dopamine is especially involved in light adaptation from darkness, the residual retina could act in triggering the turning behavior of Proteus in response to lightening.

Animals↗

Behavioural, physiological and metabolic responses to long-term starvation and refeeding in a blind cave-dwelling (Proteus anguinus) and a surface-dwelling (Euproctus asper) salamander.

The effects of long-term starvation and subsequent refeeding on haematological variables, behaviour, rates of oxygen consumption and intermediary and energy metabolism were studied in morphologically similar surface- and cave-dwelling salamanders. To provide a hypothetical general model representing the responses of amphibians to food stress, a sequential energy strategy has been proposed, suggesting that four successive phases (termed stress, transition, adaptation and recovery) can be distinguished. The metabolic response to prolonged food deprivation was monophasic in the epigean Euproctus asper (Salamandridae), showing an immediate, linear and large decrease in all the energy reserves. In contrast, the hypogean Proteus anguinus (Proteidae) displayed successive periods of glucidic, lipidic and finally lipido-proteic-dominant catabolism during the course of food deprivation. The remarkable resistance to long-term fasting and the very quick recovery from nutritional stress of this cave organism may be explained partly by its ability to remain in an extremely prolonged state of protein sparing and temporary torpor. Proteus anguinus had reduced metabolic and activity rates (considerably lower than those of most surface-dwelling amphibians). These results are interpreted as adaptations to a subterranean existence in which poor and discontinuous food supplies and/or intermittent hypoxia may occur for long periods. Therefore, P. anguinus appears to be a good example of a low-energy-system vertebrate.

Adaptation, Physiological↗

Proteus syndrome: ocular complications.

Proteus syndrome is a recently recognized hamartoneoplastic malformation syndrome of uncertain etiology and variable expression, whose cardinal manifestations are pigmented nevi, hemihypertrophy, macrodactly, lipomata, and cerebroid-gyriform configuration of the skin on the soles of the feet. The characteristic features may be present at birth but become more apparent with time. In the past this syndrome has been confused with other overgrowth disorders such as neurofibromatosis, Klippel-Trenaunay-Weber syndrome, Maffucci syndrome, and Bannayan syndrome. The ophthalmic features of the proteus syndrome require clarification. We review the ocular findings in 16 previously described cases and describe the findings unique to our patient, in particular, unilateral epibulbar and suspected posterior segment hamartomas.

Child↗