Andreas Jakob

Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O‐acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus

Staphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA, in Staphylococcus aureus. The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wild‐type strain was O‐acetylated at C6‐OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan‐specific O‐acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.

Topotecan – A Novel Topoisomerase I Inhibitor: Pharmacology and Clinical Experience

Topotecan, a water-soluble analogue of camptothecin, is a newly available cytotoxic agent which acts as an inhibitor of topoisomerase I, an enzyme necessary for DNA replication. Topotecan is a semisynthetic product derived from camptothecin, which was discovered during a National Cancer Institute cytotoxic drug screening program almost 30 years ago. It acts by forming a stable covalent complex with the DNA/topoisomerase I aggregate, the so-called ‘cleavable complex’. This process leads to breaks in the DNA strand resulting in apoptosis and cell death. Topotecan possesses a serum half-life of approximately 3 h, a high volume of distribution with high tissue uptake and a low protein binding. The chemical structure is based on a lactone ring. Topotecan undergoes reversible hydrolysis from its biologically active lactone form to the open ring inactive carboxylate form. It is also able to penetrate the intact blood-brain barrier. Since most of the agent is excreted by the kidneys, dose adjustment is necessary when renal function is impaired. In contrast, pharmacokinetic behavior is unchanged in patients with limited hepatic function. The principal toxicity of topotecan when administered at standard doses is neutropenia, but thrombocytopenia and anemia occur as well, while the nonhematological toxicities are usually mild. Alopecia is frequently observed and some patients may suffer from pronounced fatigue. Most clinical data available are based on the following schedule: 1.5 mg/m2 topotecan given as a 30-min infusion, days 1–5. There are currently only minimal data available regarding a dose–antitumor activity relationship. Other topotecan administration schedules are currently being investigated. Preclinical data suggest that continuous-infusion schedules may be a better application form in terms of both, toxicity and antitumor activity. However, clinical trials could not confirm these results to date. Results of phase II studies suggest considerable antitumor activity of single agent topotecan in small cell lung cancer and ovarian cancer patients. A randomized phase III trial of topotecan versus paclitaxel in ovarian cancer patients pretreated with cisplatin/cyclophosphamide has demonstrated that topotecan is as effective as paclitaxel in the second-line treatment of these patients. Activity of topotecan was also observed in non-small-cell lung cancer, refractory leukemias/myelodysplastic syndromes and in childhood sarcomas. Due to its unique mechanism of action and lack of cross-resistance, cisplatin, etoposide, cytarabine and paclitaxel are potential interacting partners for combination chemotherapy regimens. However, the best combination regimen as well as the optimal combination schedule have yet to be conclusively determined. The potential of topotecan in a variety of solid tumors, as well as its use in combination regimens for ovarian and small cell lung cancer is currently being investigated.

Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation.

ABSTRACTUsing two-colour flow cytometry >200 antibodies submitted to the 8th International Workshop of Human Leukocyte Differentiation Antigens (HLDA8) have been analyzed for their reactivity with resting and activated CD203c+ basophils. Four antibodies either non-reactive or weakly reactive with resting basophils exhibited an increased reactivity with basophils activated by anti-IgE-mediated cross-linking of the high affinity IgE receptor (FcεRI). These include antibodies against CD164 (WS-80160, clone N6B6 and WS-80162, clone 67D2), as well as two reagents with previously unknown specificities that were identified as CD13 (WS-80274, clone A8) and CD107a (WS-80280, clone E63-880). The activation patterns followed either the “CD203c-like” or “CD63-like” activation profile. The CD203c profile is characterized by a rapid and significant upregulation (of CD13, CD164, and CD203c), reaching maximum levels after 5-15 min of stimulation. The phosphoinositide-3-kinase (PI3K)-specific inhibitor wortmannin inhibited the upregulation of these markers whereas 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced a rapid and FcεRI-independent upregulation within 1-2 min. In the CD63 profile, maximum upregulation (of CD63 and CD107a) was detected only after 20-40 min, and upregulation by TPA reached maximum levels after 60 min. In summary, our data identify CD13, CD107a, and CD164 as novel basophil-activation antigens. Based on time kinetics of upregulation, we hypothesize that molecules of the “CD203c group” and the “CD63 group” are linked to two different mechanisms of basophil activation.

Study of enantioselective interactions between chiral drugs and serum albumin by capillary electrophoresis

The separation of the enantiomers of three basic drugs, i.e., ofloxacin, propranolol and verapamil, was achieved by affinity capillary electrophoresis (ACE), with human serum albumin (HSA) and bovine serum albumin (BSA) as chiral selectors in phosphate buffer at pH 7.4. Ofloxacin was only separated in the presence of BSA, and verapamil only with HSA, while propranolol was separated with either HSA or BSA. The effects of protein concentration and column wall adsorption on the degree of separation were investigated. Two displacers, ketoprofen and warfarin, respectively, when added to the protein containing buffer, both showed significant effects on the separation behavior. From these data it was argued that verapamil may bind to HSA at both locations known, the warfarin binding site (I) and the ketoprofen binding site (II). While with BSA, binding of ofloxacin may also occur at site I, the preferential binding site for propanolol remains controversial. A drug‐drug interaction between propranolol and ketoprofen due to opposite charges was concluded from the increase in migration time in BSA solution. The unbound concentration of verapamil enantiomers in solution in the presence of HSA, as estimated from CD‐modified capillary zone electrophoresis, was triggered not only by the HSA concentration but also by the coadditive concentration.

Inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK channel deficiency in overactive murine bladder

The large‐conductance, voltage‐dependent and Ca2+‐dependent K+ (BK) channel links membrane depolarization and local increases in cytosolic free Ca2+ to hyperpolarizing K+ outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK−/−) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time‐dependent and smooth muscle‐specific deletion of the BK channel (SM‐BK−/−) caused a more severe phenotype than displayed by constitutive BK−/− mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK−/− but not SM‐BK−/− mice, we found reduced L‐type Ca2+ current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK−/− mice was accompanied by enhanced β‐adrenoceptor/cAMP‐mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60–70% in SM‐BK−/− mice. However, the Rp isomer of adenosine‐3′,5′‐cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK−/− detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK−/− urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.

Separation of Enantiomers of Drugs by Capillary Electrophoresis with Permethyl‐gamma‐Cyclodextrin as Chiral Solvating Agent

High-throughput screening is a promising new approach in analytical chemistry. within the framework of an extended screening program (the german-chinese drug screening program), the enantioseparation of 86 drugs was investigated by capillary zone electrophoresis in the presence of the chiral solvating agent (csa) octakis-(2,3,6-tri-o-methyl)-gamma-cyclodextrin (tm-gamma-cd). by this means, 15 drugs could be separated into enantiomeric pairs. approximate measures for the degree of interaction (migration retardation factor, r-m) and for the degree of enantiomer recognition (migration separation factors, alpha(m)) revealed intriguing patterns that were compared with those found for native gamma-cyclodextrin (gamma-cd). although there is a distinct influence of the analyte structure on the electrophoretic data, interpretation remains difficult. most remarkably, permethylation of gamma-cd leads neither to a higher affinity nor to better chiral recognition, in contrast to the findings with alpha-cd.

Separation of drugs by capillary electrophoresis, Part 10. Permethyl-alpha-cyclodextrin as chiral solvating agent.

Following the German‐Chinese Drug Screening Program, 86 racemic drugs were investigated in capillary zone electrophoresis in the presence of the chiral solvating agent (CSA) hexakis‐(2,3,6‐tri‐O‐methyl)‐α‐cyclodextrin (TM‐α‐CD). Of the 86 drugs, 23 were separated into enantiomeric pairs. A comparison of the migration separation factors (αm) and the migration retardation factors (Rm) with previously published data for native α‐CD revealed that the ‘upper‐rim’ hydroxyl groups do not necessarily facilitate the recognition of the drug enantiomers by the chiral host. In contrast, an overall increase in affinity for the permethylated host led to a higher rate of successful enantiomer separations. A key substructure (4H) was identified in the analyte structure domain, with a crucial influence on the behavior of a particular drug.

Late toxicity after treatment for testicular germ cell cancer

SummaryImproved survival in testicular cancer has been accompanied by concern about long-term side effects of chemotherapy or radiotherapy. Secondary malignant neoplasia represents one of the worst possible long-term complications, leading to death in patients cured of their primary malignancy. Patients with testicular germ cell tumors appear to have a 2-fold increased risk of developing any second cancer 25–30 years after the diagnosis, resulting in a cumulative incidence of 16–23 % at that time. The risk for secondary solid tumors can be mainly attributed to radiotherapy. There is strong evidence of an increasing risk for secondary solid tumors with time since treatment. Tumor-specific analysis of the risk for second cancers revealed statistically significant excesses for stomach, pancreas, bladder, rectum, prostate, and kidney cancer, as well as for cancer of the thyroid, melanoma, sarcomas, and non-Hodgkins lymphoma. No significantly elevated risk for secondary solid tumors was observed after treatment with chemotherapy alone.The risk of secondary leukemia was associated with both radiotherapy and in particular with chemotherapy. In recent clinical surveys of patients with testicular cancer, estimates of the risk of leukemia after chemotherapy have ranged from 10- to 300-fold. An elevated risk was observed within the first two decades after diagnosis, later the risk was as expected in the normal population. Etoposide seems to be leukemogenic, especially at cumulative doses higher than 2 g/m2, although the effects of dose and schedule as well as the effects of other cytotoxic agents and radiotherapy remain to be finally clarified. Based on currently available data in patients with testicular cancer, it can be concluded that a significant elevated risk for the development of secondary leukemia exits after chemotherapy. However this risk does by far not outweigh the therapeutic benefit of etoposid-based therapy in patients with germ cell tumors.Secondary Raynauds phenomenon is the main late vascular toxicity affecting about one third of patients after curative chemotherapy for testicular cancer. Hypertension will occur in one fifths of the patients. The incidence of vascular toxicity appears to be lower following PEB-therapy compared to PVB-therapy and major vascular events seem to be rare. Other frequent symptomatic toxicities are ototoxicity and peripheral neuropathy. A major risk factor for the development of toxicity is the cumulative dose of cisplatin given. Alterations of gonadotropin levels and Leydig cell insufficiency persist in more than half of young patients cured from testicular cancer by cisplatin-based combination chemotherapy. Approximately one fourth of patients have low serum magnesium or phosphat levels, or elevated creatinine levels. These toxicities seldomly result in clinical symptoms. We conclude that 3–4 courses with bleomycin, cisplatin and etoposide in testicular cancer patients will only rarely lead to symptomatic impairment of organ functions and a decrease of quality of life.Germ cell cancers have served as a valuable model for the development of new treatment strategies contributing largely to defining the role of cisplatinum, etoposide and recently ifosfamide in medical oncology. However, germ cell cancer may also be a useful model for investigating the long term side effects of the oncological therapies. Thus, germ cell cancer is not only a “model for a curable neoplasm” (L. H. Einhorn) but can also be seen as a “model for the study of late sequelae of modern oncological therapies”.ZusammenfassungDie Verbesserung der Überlebensraten bei der Behandlung von Patienten mit Hodentumoren wird von der Sorge um mögliche Langzeitnebenwirkungen der eingesetzten Therapieformen begleitet. Eine sekundäre Neoplasie ist eine gefürchtete Langzeitnebenwirkung, die die ansonsten exzellente Prognose kurativ behandelter Patienten erheblich beeinträchtigt. Patienten mit Hodentumoren haben ein etwa 2fach erhöhtes Risiko, 25–30 Jahre nach Diagnosestellung an einer malignen Zweitneoplasie erkrankt zu sein, das entspricht einer kumulativen Inzidenz von 16–23 %. Das Risiko für sekundäre solide Tumoren ist vorwiegend mit der Strahlentherapie verbunden und scheint mit zunehmender Beobachtungsdauer anzusteigen. Für folgende Tumoren ist ein statistisch signifikant erhöhtes relatives Risiko belegt: Magenkarzinome, Pankreaskarzinome, Blasenkarzinome, kolorektale Karzinome, Prostatakarzinome, Nierenzellkarzinome, Schilddrüsenkarzinome, Melanome, Sarkome und Non-Hodgkin-Lymphome. Nach alleiniger Chemotherapie ist das Risiko für sekundäre solide Tumoren nicht signifikant erhöht. Das Risiko für sekundäre Leukämien ist auch mit der Strahlentherapie aber vorwiegend mit dem Einsatz von Chemotherapie assoziiert. Neuere klinische Analysen der Daten von Patienten mit Hodentumoren haben nach Durchführung einer Chemotherapie ein 10- bis 300faches Risiko für eine Sekundärleukämie gezeigt. Das erhöhte Risiko bezieht sich nur auf die ersten zwei Jahrzehnte nach der Diagnosestellung, danach besteht kein Unterschied zur Normalbevölkerung. Etoposid scheint leukämogen zu sein, insbesondere nach kumulativen Dosen über 2 g/m2. Die genauen Zusammenhänge mit der Dosis, der Applikationsweise, den anderen Zytostatika und der Radiotherapie sind allerdings noch nicht vollständig geklärt. Auf dem Boden der derzeit verfügbaren Daten von Patienten mit Hodentumoren ist festzustellen, daß nach einer chemotherapeutischen Behandlung ein signifikant erhöhtes Risiko für eine Sekundärleukämie besteht. Bei gleichzeitiger Abwägung der hohen Heilungsrate ist das Risiko für den individuellen Patienten gering, aber nicht vollständig zu vernachlässigen. Das sekundäre Raynaud-Syndrom stellt die wesentliche vaskuläre Spättoxizität dar und betrifft etwa ein Drittel der Patienten nach kurativer Chemotherapie des Hodentumors. Bei einem fünftel der Patienten tritt eine arterielle Hypertonie auf. Es ist allerdings zu erwarten, daß die Häufigkeit dieser Nebenwirkungen durch Einsatz des PEB-Regimes (im Vergleich zum PVB-Regime) in Zukunft sinken werden. Schwere vaskuläre Komplikationen scheinen nur sehr selten aufzutreten. Weitere häufige Toxizitäten sind Ototoxizität und periphere Neuropathie. Einer der wichtigsten Risikofaktoren für das Auftreten dieser Toxizitäten ist die kumulativ applizierte Cisplatindosis. Bei mehr als der Hälfte der jungen Patientenpopulation ließen sich, nach erfolgreicher cisplatin-basierten Kombinationschemotherapie, persistierende Veränderungen der Gonadotropinspiegel und eine Leydigzellinsuffizienz nachweisen. Etwa ein viertel der Patienten wiesen erniedrigte Magnesium- oder Phosphatwerte auf, oder hatten einen erhöhten Kreatininwert im Serum. Diese Toxizitäten führen selten zu klinischen Symptomen. Zusammengefaßt läßt sich derzeit feststellen, daß 3-4 Serien einer Therapie mit Bleomycin, Cisplatin und Etoposid bei Patienten mit Hodentumoren nur selten zu symptomatischen Störungen der Organfunktionen und zu einer Beeinträchtigung der Lebensqualität führen. Die vorliegende Übersicht zeigt, daß das „Modell Hodentumor“ nicht nur die Therapieentwicklung, bei der es ja entscheidend zur Definition der Rolle von Cisplatin, Etoposid und in neuerer Zeit von Ifosfamid in der klinischen Onkologie beigetragen hat, sondern auch für die Untersuchung der Langzeitfolgen dieser eingesetzten Therapien von breiter klinischer Relevanz ist. Somit könnte der maligne Hodentumor nicht nur „a model for a curable neoplasm“ (LH. Einhorn) sondern auch „a model for the study of late sequelae of modern oncological therapies“ darstellen.

Transient state of chiral recognition in a binary mixture of cyclodextrins in capillary electrophoresis

The transient state (as the defined point where no enantioseparation is obtained in a dual chiral selector system) of chiral recognition of aminoglutethimide in a binary mixture of neutral cyclodextrins (CDs) was studied by capillary electrophoresis (CE). The following three dual selector systems were used: alpha-cyclodextrin (alpha-CD) and beta-cyclodextrin (beta-CD); alpha-CD and heptakis(di-O-methyl-beta-cyclodextrin) (DM-beta-CD); alpha-CD and heptakis(tri-O-methyl-beta-cyclodextrin) (TM-beta-CD). The S-(-) enantiomer of the analyte was more strongly retained in the presence of either alpha-CD or TM-beta-CD at pH 2.5, 100 mM phosphate buffer, while the R-(+) enantiomer was more strongly retained in the presence of either beta-CD or DM-beta-CD. In the more simple case, the elution order is invariably kept if the enantiomers have the same elution order in either one of the two hosts of the binary mixture. In contrast, the elution order may be switched by varying the concentration ratio of two hosts that produce opposite elution order for this particular analyte. In such a dual selector system, the enantioselectivity will disappear at the transient state at a certain ratio of host1:host2. Moreover, the migration times of the two enantiomers with host, alone (diluted in buffer) is approximately equal to the migration times at the corresponding concentration of host2 alone (diluted in buffer), where the ratio of concentrations of host1:host2 is the same as in the binary mixture at the transient state. As found by nuclear magnetic resonance experiments, the analyte is forming a 1:1 complex with either one of the CDs applied. From this finding, a theoretical model based on the mobility difference of the two enantiomers was derived that was used to simulate the transient state.

Optimization and parameter study for chiral separation by capillary electrophoresis

Orthogonal design and uniform design were used for the optimization of separation of enantiomers using 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD) as a chiral selector by capillary zone electrophoresis, The concentration of DM-beta-CD, buffer pH, running voltage, and capillary temperature were selected as variable parameters, their different effects on peak resolution were studied by the design methods. It was concluded that orthogonal design offers a rapid and efficient means for testing the importance of individual parameters and for determining the optimum operating conditions. However, for a large number of both factors and levels, uniform design is more efficient, The effect of addition of methanol and citric acid buffer on the separation of enantiomers was also examined.