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The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.
Christian Müller; Phil Lukas; Dana Sponholz; Jan-Peter Hildebrandt. The hirudin-like factors HLF3 and HLF4—hidden hirudins of European medicinal leeches. Parasitology Research 2020, 119, 1767 -1775.
AMA StyleChristian Müller, Phil Lukas, Dana Sponholz, Jan-Peter Hildebrandt. The hirudin-like factors HLF3 and HLF4—hidden hirudins of European medicinal leeches. Parasitology Research. 2020; 119 (6):1767-1775.
Chicago/Turabian StyleChristian Müller; Phil Lukas; Dana Sponholz; Jan-Peter Hildebrandt. 2020. "The hirudin-like factors HLF3 and HLF4—hidden hirudins of European medicinal leeches." Parasitology Research 119, no. 6: 1767-1775.
The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anti‐coagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N‐terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF‐variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.
Christian Müller; Phil Lukas; Michel Böhmert; Jan‐Peter Hildebrandt. Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants. FEBS Letters 2019, 594, 841 -850.
AMA StyleChristian Müller, Phil Lukas, Michel Böhmert, Jan‐Peter Hildebrandt. Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants. FEBS Letters. 2019; 594 (5):841-850.
Chicago/Turabian StyleChristian Müller; Phil Lukas; Michel Böhmert; Jan‐Peter Hildebrandt. 2019. "Hirudin or hirudin‐like factor ‐ that is the question: insights from the analyses of natural and synthetic HLF variants." FEBS Letters 594, no. 5: 841-850.
Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.
Phil Lukas; Robert Wolf; Bernhard H. Rauch; Jan-Peter Hildebrandt; Christian Müller. Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different. Parasitology Research 2019, 118, 2223 -2233.
AMA StylePhil Lukas, Robert Wolf, Bernhard H. Rauch, Jan-Peter Hildebrandt, Christian Müller. Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different. Parasitology Research. 2019; 118 (7):2223-2233.
Chicago/Turabian StylePhil Lukas; Robert Wolf; Bernhard H. Rauch; Jan-Peter Hildebrandt; Christian Müller. 2019. "Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different." Parasitology Research 118, no. 7: 2223-2233.
Interaction of Staphylococcus aureus alpha-toxin (hemolysin A, Hla) with eukaryotic cell membranes is mediated by proteinaceous receptors and certain lipid domains in host cell plasma membranes. Hla is secreted as a 33 kDa monomer that forms heptameric transmembrane pores whose action compromises maintenance of cell shape and epithelial tightness. It is not exactly known whether certain membrane lipid domains of host cells facilitate adhesion of Ha monomers, oligomerization, or pore formation. We used sphingomyelinase (hemolysin B, Hlb) expressed by some strains of staphylococci to pre-treat airway epithelial model cells in order to specifically decrease the sphingomyelin (SM) abundance in their plasma membranes. Such a pre-incubation exclusively removed SM from the plasma membrane lipid fraction. It abrogated the formation of heptamers and prevented the formation of functional transmembrane pores. Hla exposure of rHlb pre-treated cells did not result in increases in [Ca2+]i, did not induce any microscopically visible changes in cell shape or formation of paracellular gaps, and did not induce hypo-phosphorylation of the actin depolymerizing factor cofilin as usual. Removal of sphingomyelin from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of Staphylococcus aureus alpha-toxin.
Sabine Ziesemer; Nils Möller; Andreas Nitsch; Christian Müller; Achim G. Beule; Jan-Peter Hildebrandt. Sphingomyelin Depletion from Plasma Membranes of Human Airway Epithelial Cells Completely Abrogates the Deleterious Actions of S. aureus Alpha-Toxin. Toxins 2019, 11, 126 .
AMA StyleSabine Ziesemer, Nils Möller, Andreas Nitsch, Christian Müller, Achim G. Beule, Jan-Peter Hildebrandt. Sphingomyelin Depletion from Plasma Membranes of Human Airway Epithelial Cells Completely Abrogates the Deleterious Actions of S. aureus Alpha-Toxin. Toxins. 2019; 11 (2):126.
Chicago/Turabian StyleSabine Ziesemer; Nils Möller; Andreas Nitsch; Christian Müller; Achim G. Beule; Jan-Peter Hildebrandt. 2019. "Sphingomyelin Depletion from Plasma Membranes of Human Airway Epithelial Cells Completely Abrogates the Deleterious Actions of S. aureus Alpha-Toxin." Toxins 11, no. 2: 126.
Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus alpha-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity which are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeleton. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin depolymerizing factor cofilin. We used recombinant wild-type Hla (rHla) as well as a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla-pores in the host cell membrane. Formation of functional transmembrane pores induced hypo-phosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of PAK- and LIMK activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.
Sabine Ziesemer; Ina Eiffler; Alfrun Schönberg; Christian Müller; Falko Hochgräfe; Achim G. Beule; Jan-Peter Hildebrandt. Staphylococcus aureusα-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells. American Journal of Respiratory Cell and Molecular Biology 2018, 58, 482 -491.
AMA StyleSabine Ziesemer, Ina Eiffler, Alfrun Schönberg, Christian Müller, Falko Hochgräfe, Achim G. Beule, Jan-Peter Hildebrandt. Staphylococcus aureusα-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells. American Journal of Respiratory Cell and Molecular Biology. 2018; 58 (4):482-491.
Chicago/Turabian StyleSabine Ziesemer; Ina Eiffler; Alfrun Schönberg; Christian Müller; Falko Hochgräfe; Achim G. Beule; Jan-Peter Hildebrandt. 2018. "Staphylococcus aureusα-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells." American Journal of Respiratory Cell and Molecular Biology 58, no. 4: 482-491.
Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla). This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins) activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L), which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.
Romina Baaske; Mandy Richter; Nils Möller; Sabine Ziesemer; Ina Eiffler; Christian Müller; Jan-Peter Hildebrandt. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin. Toxins 2016, 8, 365 .
AMA StyleRomina Baaske, Mandy Richter, Nils Möller, Sabine Ziesemer, Ina Eiffler, Christian Müller, Jan-Peter Hildebrandt. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin. Toxins. 2016; 8 (12):365.
Chicago/Turabian StyleRomina Baaske; Mandy Richter; Nils Möller; Sabine Ziesemer; Ina Eiffler; Christian Müller; Jan-Peter Hildebrandt. 2016. "ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin." Toxins 8, no. 12: 365.
Haematophagous leeches express a broad variety of bioactive factors that are released from the salivary gland cells into the wound of a host during feeding. Among these, hirudin is probably the best studied factor and, moreover, the only one that has successfully made the transition from nature to clinical use. Many components of the leech saliva still remain either poorly characterized or yet completely unknown. Only recently, a new class of leech-derived factors has been discovered in Hirudo medicinalis, the hirudin-like factors (HLFs). HLFs comprise typical structural features of hirudin but lack others. We were able to verify the expression of HLFs not only in two additional species of the genus Hirudo, but also in Hirudinaria manillensis. Various phylogenetic analyses based on gene and protein sequences support a sister group relationship between hirudins and HLFs. Although potential molecular targets of HLFs remain unknown, the presence of multiple isoforms in individual leeches of different genera points to key functions in the regulation of several processes associated with the blood-sucking life style of leeches.
Christian Müller; Martin Haase; Sarah Lemke; Jan-Peter Hildebrandt. Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships. Parasitology Research 2016, 116, 313 -325.
AMA StyleChristian Müller, Martin Haase, Sarah Lemke, Jan-Peter Hildebrandt. Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships. Parasitology Research. 2016; 116 (1):313-325.
Chicago/Turabian StyleChristian Müller; Martin Haase; Sarah Lemke; Jan-Peter Hildebrandt. 2016. "Hirudins and hirudin-like factors in Hirudinidae: implications for function and phylogenetic relationships." Parasitology Research 116, no. 1: 313-325.
Blood-sucking leeches like the medicinal leech, Hirudo medicinalis, have been used for medical purposes since ancient times. During feeding, medicinal leeches transfer a broad range of bioactive substances into the host’s wound to prevent premature hemostasis and blood coagulation. Hirudin is probably the best known of these substances. Despite its long history of investigation, recombinant production and clinical use, there still exist conflicting data regarding the primary structure of hirudin. Entirely unclear is the potential biological significance of three different subtypes and many isoforms of hirudins that have been characterized so far. Furthermore, there is only incomplete information on their cDNA sequences and no information at all on gene structures and DNA sequences are available in the databases. Our efforts to fill these gaps revealed the presence of multiple hirudin-encoding genes in the genome of Hirudo medicinalis. We have strong evidence for the expression of all three subtypes of hirudin within individual leeches and for the expression of additional hirudins or hirudin-like factors that may have different biological functions and may be promising candidates for new drugs.
Christian Müller; Katharina Mescke; Stephanie Liebig; Hala Mahfoud; Sarah Lemke; Jan-Peter Hildebrandt. More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis. Molecular Genetics and Genomics 2015, 291, 227 -240.
AMA StyleChristian Müller, Katharina Mescke, Stephanie Liebig, Hala Mahfoud, Sarah Lemke, Jan-Peter Hildebrandt. More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis. Molecular Genetics and Genomics. 2015; 291 (1):227-240.
Chicago/Turabian StyleChristian Müller; Katharina Mescke; Stephanie Liebig; Hala Mahfoud; Sarah Lemke; Jan-Peter Hildebrandt. 2015. "More than just one: multiplicity of Hirudins and Hirudin-like Factors in the Medicinal Leech, Hirudo medicinalis." Molecular Genetics and Genomics 291, no. 1: 227-240.
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h. Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1. Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH. Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
Anne-Katrin Rohlfing; Karoline Trescher; Juliane Hähnel; Christian Müller; Jan-Peter Hildebrandt. Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes. Frontiers in Physiology 2013, 4, 139 .
AMA StyleAnne-Katrin Rohlfing, Karoline Trescher, Juliane Hähnel, Christian Müller, Jan-Peter Hildebrandt. Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes. Frontiers in Physiology. 2013; 4 ():139.
Chicago/Turabian StyleAnne-Katrin Rohlfing; Karoline Trescher; Juliane Hähnel; Christian Müller; Jan-Peter Hildebrandt. 2013. "Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes." Frontiers in Physiology 4, no. : 139.