Boris Krajnc je bil prvi habilitirani učitelj ljubljanske univerze za področje biokemije – imenovanje v naziv docenta je datirano s 7. januarjem 1946. Vendar se je Krajnčeva življenjska pot zelo ...hitro končala, saj so ga 27. oktobra 1947 aretirali in 26. aprila 1948 na montiranem dachauskem procesu obsodili na smrt. Ustrelili naj bi ga 12. maja 1948, starega 34 let. Krajnčeva življenjska zgodba je tesno povezana z delom več slovenskih kemikov, ki so bili kot zaporniki v koncentracijskem taborišču Dachau izbrani za tehnično pomoč pri izvedbah poskusov na ljudeh ali pri delu v kliničnem laboratoriju. Na povojnih dachauskih procesih v Ljubljani je bilo skupaj obsojenih 8 kemikov, od teh jih je bilo 5 obsojenih na smrt in ustreljenih. V kasnejših presojah dachauskih procesov se je izkazalo, da so bile obtožnice skonstruirane, priznanja pa pridobljena s krutimi zasliševalskimi metodami, zato so bile vse sodbe razveljavljene. Po emigraciji encimatika Richarda Klemna ter pregonu Maksa Samca in Marte Blinc z univerze je bil Boris Krajnc tisti, ki naj bi skrbel za biokemijo na Tehniški fakulteti. Po njegovi smrti je nekaj let biokemijo predaval Krajnčev mlajši kolega Dušan Stucin z Medicinske fakultete, potem pa biokemije več let ni bilo v kemijskem kurikulumu.
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Ob 100-letnici rojstva znane kristalografinje Rosalind Franklin (1920-1958) članek opisuje njene stike s slovenskimi in hrvaškimi kemiki v letih 1952-1955, predvsem z Dušanom Hadžijem in Katarino ...Kranjc. Ugotovil sem, da je najbolj znana planinska fotografija Rosalind Franklin nastala v Julijskih Alpah verjetno leta 1952 in ne na Norveškem konec 30-tih let, kot je veljalo doslej. Na Triglav pa se je vzpela julija 1954 ali septembra 1955 in ne leta 1952, kot piše v njeni biografiji.
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Richard Klemen was the first teacher of enzymology at the University of Ljubljana. His early career in Ljubljana ended in January 1942 when he moved to Vienna, Austria. During the war he conducted ...experiments that led him to describe the so-called Hofmann-Klemen effect in clay. Later he was a research assistant and titular associate professor in the field of biochemical technology at the Vienna Technical University and finally a lecturer at the University of Natural Resources in Vienna. His life is an interesting example of a scientist and educator whose Gottscheer German origin would probably prevent him from continuing his career in post-war Yugoslavia. At the same time, he did not achieve in Austria the positions and status that his former colleagues and students had achieved in Slovenia. Although he was almost forgotten, he remains important as the first trained enzymologist and teacher of enzymology in Slovenia. This article also presents his full bibliography.
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Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced
...toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium
sp. PCC 6803.
We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in
and
sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target.
With the new vector we observed a significantly higher GFP expression in
and
sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both
and
sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
The bloom-forming cyanobacterium
is known for its global distribution and for the production of toxic compounds. In the genome of
PCC 7806, we discovered that the gene coding for MaOC1, a caspase ...homolog protease, is followed by a toxin-antitoxin module, flanked on each side by a direct repeat. We therefore investigated their possible interaction at the protein level. Our results suggest that this module belongs to the ParE/ParD-like superfamily of type II toxin-antitoxin systems. In solution, the antitoxin is predominantly alpha-helical and dimeric. When coexpressed with its cognate toxin and isolated from
, it forms a complex, as revealed by light scattering and affinity purification. The active site of the toxin is restricted to the C-terminus of the molecule. Its truncation led to normal cell growth, while the wild-type form prevented bacterial growth in liquid medium. The orthocaspase MaOC1 was able to cleave the antitoxin so that it could no longer block the toxin activity. The most likely target of the protease was the C-terminus of the antitoxin with two sections of basic amino acid residues.
cells in which MaOC1 was expressed simultaneously with the toxin-antitoxin pair were unable to grow. In contrast, no effect on cell growth was found when using a proteolytically inactive MaOC1 mutant. We thus present the first case of a cysteine protease that regulates the activity of a toxin-antitoxin module, since all currently known activating proteases are of the serine type.
Orthocaspases are prokaryotic caspase homologs - proteases, which cleave their substrates after positively charged residues using a conserved histidine - cysteine (HC) dyad situated in a catalytic ...p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis (orders Rhizobiales, Rhodobacterales, and Rhodospirillales in Alphaproteobacteria) or oxygenic photosynthesis (all sequenced cyanobacteria, except
,
, and
). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria
and
comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.
Cyanobacteria are an important group of microorganisms displaying a range of morphologies that enable phenotypic differentiation between the major lineages of cyanobacteria, often to the genus level, ...but rarely to species or strain level. We focused on the unicellular genus Synechocystis that includes the model cyanobacterial strain PCC 6803. For 11 Synechocystis members obtained from cell culture collections, we sequenced the variable part of the 16S rRNA-encoding region and the 16S - 23S internally transcribed spacer (ITS), both standardly used in taxonomy. In combination with microscopic examination we observed that 2 out of 11 strains from cell culture collections were clearly different from typical Synechocystis members. For the rest of the samples, we demonstrated that both sequenced genomic regions are useful for discrimination between investigated species and that the ITS region alone allows for a reliable differentiation between Synechocystis strains.
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At the University of Ljubljana, Boris Krajnc was the first habilitated teacher to cover the subject of biochemistry: he was appointed on January 5, 1946. However, Krajnc’s life ended abruptly. He was ...arrested on October 17, 1947 and sentenced to death on April 26, 1948 in the mounted Dachau Trial in Ljubljana. He was reportedly shot on May 12, 1948, at the age of 34. The life story of Boris Krajnc is closely linked to the work of several Slovenian chemists who were selected as prisoners at Dachau Concentration Camp to provide technical assistance in human experiments or in the clinical laboratory of the camp hospital. In the post-war period Dachau Trials in Ljubljana, a total of 8 chemists were convicted, 5 of whom were sentenced to death and executed by firing squad. In later evaluations of Dachau Trials it turned out that the charges were constructed and the confessions were obtained by cruel interrogation methods, therefore all judgements were annulled. After the emigration of the enzymologist Richard Klemen (1942) and the expulsion of Maks Samec and Marta Blinc from the university (1945), Boris Krajnc was to supervise biochemistry at the Technical Faculty. After his death, the lecturer of biochemistry was Dušan Stucin from the Medical Faculty. Later, biochemistry was absent from the chemistry curriculum for several years.
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In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can ...have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacteriumSynechocystissp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA fromAnabaenacombined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising thenucAgene fused to a variant of thecopMpromoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring ofSynechocystisTA pairsssr1114/slr0664andslr6101/slr6100for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including anrsBvariant, were characterized by beta-galactosidase reporter assay.
Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine ...antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.
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