Morphological study of the reversion to bacillary form, of Bacillus megaterium protoplasts.

Protoplasts of Bacillus megaterium readily reverted to bacillary form in liquid media and when plated in a soft-agar layer onto the surface of appropriate agar media. Three phases of the reversion sequence could be differentiated by phase contrast microscopy: (i) increase in size of the individual protoplasts, (ii) non oriented division of the protoplasts and (iii) outgrowth of the bacillary forms. With time-lapse photomicrography, reversion sequences of single protoplasts were demonstrated.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.     

The peroxisomal receptor Pex19p forms a helical mPTS recognition domain

The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C‐terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured α‐helical domain binds PMP‐targeting signal (mPTS) sequences with about 10 μM affinity. Removal of a conserved N‐terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS‐binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N‐terminal and C‐terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo‐targeting signal recognition and additional functions.     

Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine

Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a β-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants.     

Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.Xenorhabdus and Photorhabdus bacteria are highly virulent, fatal pathogens for insects. Phylogenetically, they are sister genera in the family Enterobacteriaceae (3, 4). There are some differences between Xenorhabdus and Photorhabdus in their biology (e.g., light production), and they also differ in their interaction with their symbiotic nematode partners, which are in the Steinernematidae and Heterorhabditidae genera, respectively (8, 9). At the same time, they also have several properties in common. For example, due to their similar strategy of infection, their entrance into the hemocoel is absolutely dependent on the invasion of insects by their symbiotic nematode partners. An interesting feature of both genera is that they have two phenotypic (form) variants, primary and secondary (9). The primary form is natural, while the secondary form can be observed (generated) mostly in the laboratory. They differ in, for example, antibiotic production, outer membrane proteins, and cell surface structures (fimbriae and flagellae [23], symbiotic capabilities with nematode partners, and exoenzyme production [9]). The secondary form variants were found, with nonbiochemical detection methods, to produce less or no proteolytic activity compared to the primary phenotypic variants (see references 9 and 23 and references therein). The high pathogenicity makes Xenorhabdus and Photorhabdus good model organisms of infection, which can be exploited—by studying the function of their virulence factors—for the investigation of the immune system of insects and the mechanisms the pathogens use to cope with the immune defense of hosts. The comparative analysis of these bacterial partners provides an opportunity to study the question of how similar the infection mechanisms can be at the molecular level of two evolutionarily different insect pathogen bacterium-nematode complexes that, at the same time, have similar infection strategies.Of the virulence factors, we have been interested in secreted proteases that may be used by the pathogens during the first stage of infection in the penetration of the tissues of host or in the suppression of its immune response. The secretion and biochemistry of these enzymes are better studied in Photorhabdus, where four secreted proteases could be detected in a screen of 20 strains by a combination of five methods (15). The earliest secreted Photorhabdus protease is PrtA peptidase, a metzincin in the M10B family of serralysins. The others are PhpC (Photorhabdus protease C), which belongs to the M4 metallopeptidase family of thermolysin-like proteases, OpdA, a collagen peptidase in the family of thimet oligopeptidases and PhpD, a furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY)-cleaving enzyme, the identity of which is still unknown. In contrast, although a number of Xenorhabdus strains were tested for proteolytic activity with simple bacteriological plate assays (2, 25), only one (Xenorhabdus nematophila) was investigated by a biochemical detection method of protease activities, zymography. Two activities have been found by this method, and one of these activities has been partially characterized (5).As an approach to establish the similarity between Xenorhabdus and Photorhabdus in the mechanism of infection regarding the type and role of proteolytic enzymes, we investigated 15 Xenorhabdus strains for the secretion of proteases employing the same five detection methods that we had previously used for Photorhabdus strains. Two of the strains (Xenorhabdus nematophila AN6 and Xenorhabdus cabanillassii RIO-HU) were represented with their phenotypic variant pairs.     

Correlation of iron content, spectral forms of chlorophyll and chlorophyll-proteins in iron deficient cucumber (Cucumis sativus)

Leaves and chloroplast suspensions of severely and slightly iron deficient cucumber ( Cucumis sativus L.) plants were characterized by low-temperature fluorescence emission spectroscopy and Deriphat polyacrylamide gel electrophoresis. The emission spectra of the chloroplast suspensions were resolved into Gaussian components and those changes induced by iron deficiency were related to the variations in the chlorophyll-protein pattern. The symptoms described with these methods were also correlated with the iron content of the leaves. It was concluded that the lack of physiologically active iron caused a relative decrease of photosystem I (PSI) and light harvesting complex I (LHCI), together with the long wavelength fluorescence, especially the 740 nm Gaussian component, and. to a much lesser extent, of the photosystem II (PSII) core complexes (relative increase of 685, 695 nm components). However, the relative decrease in the amount of light harvesting complex II (LHCII) was followed by a relative increase in its fluorescence band at 680 nm, showing that energy transfer from LHCII to core complex II (CCII) was partly disturbed. Thus iron deficiency affected the photosynthetic apparatus in a complex way: it decreased the synthesis of chlorophylls (Chls) and influenced the expression and assembly of Chl-binding proteins.     

Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study

Brain Cell Biology - An antiserum, to substance P has been used to study the neostriatum of rats which has received intracerebral injections of colchicine. Both cell bodies and nerve fibres were...