Phylogenetic aspects of the sulfate assimilation genes from Thalassiosira pseudonana

Diatoms are unicellular algae responsible for approximately 20 % of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids. In the last years the interest on unicellular algae increased. On the one hand assessments suggest that diatom-mediated export production can influence climate change through uptake and sequestration of atmospheric CO₂. On the other hand diatoms are in focus because they are discussed as potential producer of biofuels. To follow the one or other idea it is necessary to investigate the diatoms biochemistry in order to understand the cellular regulatory mechanisms. The sulfur assimilation and methionine synthesis pathways provide S-containing amino acids for the synthesis of proteins and a range of metabolites such as dimethylsulfoniopropionate (DMSP) in order to provide basic metabolic precursors needed for the diatoms metabolism. To obtain an insight into the localization and organization of the sulfur metabolism pathways, the genome of Thalassiosira pseudonana—a model organism for diatom research—might help to understand the fundamental questions on adaptive responses of diatoms to dynamic environmental conditions such as nutrient availability in a broader context.     

One-pot efficient synthesis of N α-urethane-protected β- and γ-amino acids

1-[(4-Methylphenyl)oxy]pyrrolidine-2,5-dione and 1-[(4-methylphenyl)oxy]piperidine-2,6-dione react in a Lossen-type reaction with primary alcohols in the presence of triethylamine to furnish corresponding N ^α-urethane-protected β-alanine and γ-aminopropionic acid (GABA), respectively, with excellent yields and purities, in an essentially “one-pot” procedure.     

Organization of two-component monomolecular layers formed with dipalmitoylphosphatidylcholine and the carotenoid pigment,canthaxanthin

Canthaxanthin is a carotenoid pigment of physiological importance owing to potential modulation of the dynamic and structural properties of biomembranes. The effect of canthaxanthin on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of monomolecular layer technique, FTIR spectroscopy and linear dichroism-FTIR. The specific molecular areas of the two-component monomolecular layers of canthaxanthin-DPPC show pronounced underadditivity in the concentration range below 2 mol% carotenoid with respect to the lipid, corresponding to the monomeric organization of the pigment. Additionally, the analysis of the FTIR spectra of the two-component monolayers deposited to the solid support shows that organization of the carotenoid in the lipid monolayer is governed primarily by van der Waals interactions between the pigment chromophore and lipid alkyl chains. This interaction is responsible for an ordering effect of canthaxanthin with respect to lipids. Analysis of FTIR spectra of two-component monolayers suggests the possibility of hydrogen bonding between the lipid polar headgroups and the keto groups of canthaxanthin via water bridges.     

The Unique hmuY Gene Sequence as a Specific Marker of Porphyromonas gingivalis

Porphyromonas gingivalis, a major etiological agent of chronic periodontitis, acquires heme from host hemoproteins using the HmuY hemophore. The aim of this study was to develop a specific P. gingivalis marker based on a hmuY gene sequence. Subgingival samples were collected from 66 patients with chronic periodontitis and 40 healthy subjects and the entire hmuY gene was analyzed in positive samples. Phylogenetic analyses demonstrated that both the amino acid sequence of the HmuY protein and the nucleotide sequence of the hmuY gene are unique among P. gingivalis strains/isolates and show low identity to sequences found in other species (below 50 and 56%, respectively). In agreement with these findings, a set of hmuY gene-based primers and standard/real-time PCR with SYBR Green chemistry allowed us to specifically detect P. gingivalis in patients with chronic periodontitis (77.3%) and healthy subjects (20%), the latter possessing lower number of P. gingivalis cells and total bacterial cells. Isolates from healthy subjects possess the hmuY gene-based nucleotide sequence pattern occurring in W83/W50/A7436 (n = 4), 381/ATCC 33277 (n = 3) or TDC60 (n = 1) strains, whereas those from patients typically have TDC60 (n = 21), W83/W50/A7436 (n = 17) and 381/ATCC 33277 (n = 13) strains. We observed a significant correlation between periodontal index of risk of infectiousness (PIRI) and the presence/absence of P. gingivalis (regardless of the hmuY gene-based sequence pattern of the isolate identified [r = 0.43; P = 0.0002] and considering particular isolate pattern [r = 0.38; P = 0.0012]). In conclusion, we demonstrated that the hmuY gene sequence or its fragments may be used as one of the molecular markers of P. gingivalis.     

Bactericidal effect of photodynamic therapy against methicillin-resistant Staphylococcus aureus strain with the use of various porphyrin photosensitizers

Photodynamic therapy (PDT) is based on photosensitizers activated by light of appropriate wavelength. Their activation leads to generation of singlet oxygen and free radicals responsible for the cytotoxic effect. The aim of this project was to compare the bactericidal effect of PDT using different porphyrin photosensitizers against a methicillin-resistant Staphylococcus aureus strain. Exogenous sensitizers (protoporphyrin IX and newly synthesized derivative, protoporphyrin diarginate) induced a 3 log10-unit reduction in bacterial viable counts. With the use of endogenous, ALA-induced porphyrins, a 1.6 log10-unit reduction was obtained. The sensitizers tested executed their antibacterial activity with no essential change in the antibiotic resistance pattern of the studied strain.     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

CD40L and IL-4 stimulation of acute lymphoblastic leukemia cells results in upregulation of mRNA level of FLICE--an important component of apoptosis

The use of cancer vaccines based on dendritic cells (DC) presenting tumor antigens can be a promising tool in the treatment of leukemia. The functional characteristics of leukemia derived DC is still to be elucidated. CD40 promotes survival, proliferation and differentiation of normal B cells. CD40 triggering was used to enhance the poor antigen-presenting capacity of leukemic B-cells. Since it is still unclear whether CD40 ligation drives neoplastic B-cells to apoptosis or not, we assessed the mRNA expression of FLICE, FAS, FADD and TRADD - important components of apoptosis machinery, using real-time PCR in acute lymphoblastic leukemia cells before and after CD40 and IL-4 stimulation. ALL cells stimulated with CD40L/IL-4 expressed dendritic cell phenotype at mRNA and protein levels (upregulation of main costimulatory and adhesion molecules noted in real-time RT PCR and flow cytometry); they also expressed higher amounts of mRNA for FLICE, TRADD and FADD after CD40L/IL-4 stimulation. However differences statistically significant comparing cells cultured with CD40L/IL-4 and medium alone regarded only FLICE. Concluding, we showed upregulation of important elements of apoptosis at mRNA level in ALL cells after CD40 ligation.