Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model.

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is 'ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a moderate increase in apoptosis, as well as dephosphorylation of ERK1/2 and AKT/PKB. These data clearly indicate ERBB2 dependence. Therefore, a high sensitivity to trastuzumab may be suspected. Surprisingly, trastuzumab caused a much weaker effect compared to ATc-induced ERBB2 downregulation, although a decrease in ERBB2 membrane localisation was induced. Only a slight decrease in proliferation and a weak transient increase in apoptosis were observed. Interestingly, tumours responded to trastuzumab by a sharp fivefold increase in phosphorylated AKT/PKB as well as a 3.5- and 5.3-fold increase in AKT1 and AKT2 mRNA levels, respectively. In conclusion, 'ERBB2 dependence' is not sufficient to define trastuzumab-responsive tumours. The suboptimal effect of trastuzumab compared to the maximally possible effect induced by ATc demonstrates a high potential for improved ERBB2 blocking therapies.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

[Treatment specificity in chronic-back pain: is optimized rehabilitation allocation enough?]. / Behandlungsspezifik beim chronischen Rückenschmerz: Ist die optimierte Rehabilitationszuweisung ausreichend?

The multidimensional and heterogeneous etiogenesis of chronic low back pain requires multimodal as well as specific treatment programmes. Aim of the present study was to increase the specificity of treatment already at the interface "family health care - assigning institution - rehabilitation clinic" by supporting the assignment to orthopaedic or psychosomatic rehabilitation through extensive diagnostics and deriving a patient's profile of risk factors in the forefield of rehabilitation. In a prospective randomized control group design the rehabilitation success in 309 persons (age 22 - 58) suffering from chronic low back pain was compared on risk factor specific vs. conventional assignment. Between 22 % and 27 % of the assignments following conventional criteria have to be considered as false. On the other hand, rehabilitation success did not turn out to have been increased by specific assignment. In conclusion, implications for more far-reaching change in treatment programmes within and subsequent to stationary rehabilitation are discussed.

Urea-induced denaturation of human phenylalanine hydroxylase.

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of the fusion partner, the effects of increasing urea concentrations on enzyme activity, aggregation, unfolding, and refolding were examined. At pH 7.50, purified human phenylalanine hydroxylase is transiently activated in the presence of 0-4 M urea but slowly inactivated at higher denaturant concentrations. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phenylalanine (L-Phe) shifts the transition midpoint of the first transition from 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant differences in dDeltaG(D)/d[urea] (m(D) values) were observed for the first denaturation transition. In the absence of substrate, a high rate of non-covalent aggregation was observed for the enzyme in the presence of 1-4 M urea. All three tryptophan residues in the enzyme (Trp-120, Trp-187, and Trp-326) were mutated to phenylalanine, either as single mutations or in combination, in order to identify the residues involved in the spectroscopic transitions. A gradual dissociation of the native tetrameric enzyme to increasingly denatured dimeric and monomeric forms was demonstrated by size exclusion chromatography in the presence of denaturants.

Secretion of acid proteinases by different species of the genus Candida.

Strains of several Candida species that are isolated from humans aside of C. albicans have been screened for secretion of acid proteinases. A third of the strains of C. tropicalis was strongly proteolytic, other strains of this species were moderately or virtually nonproteolytic; the variety of activities resembled pattern found among strains of C. albicans (32). Strains of C. parapsilosis produced 24% of the proteolytic activity of strongly proteolytic strains of C. tropicalis. The proteolytic activity of C. pseudotropicalis, C. krusei, and C. glabrata accounted for 7.5, 2.0, and 0.8% respectively. Four strains of C. guilliermondii developed strain-specific proteolysis after prolonged cultivation only. Five proteinases from strains of C. tropicalis cross reacted immunologically as did five proteinases from C. albicans. Interspecific cross reaction, though, was not observed when certain sera from rabbits were employed.

[Homogenization of the specimens for culturing mycobacteria by means of the detergents Präwozell N-BX and ditalan E (author's transl)]. / Uber die Verwendung der ionenaktiven Netzmittel Präwozell N-BX und Ditalan E als Vorbehandlungsmittel zum kulturellen Nachweis von Mykobakterien

In cooperation of four laboratories the efficiency of the two detergents Präwozell N-BX (Nekal BX) and Ditalan E used for homogenization of diagnostic specimens for cultural investigations on tuberculosis was compared. The pretreatment of sputum specimens was done according to the directions given in "DAB 7 (D.L.) - DDR" [German Pharmacopocia 7(Diagnostic laboratory methods) GDR]. Percentage of positive cultures, time of growth and rate of contamination were evaluated. Comparative investigations with both methods using the same sputum specimens were performed with 2117 samples. 245 cultures were positive in all, 223 (91,0%) of them after homogenization by means of Präwozell N-BX and 205 (83,7%) pretreated with Ditalan E. Out of 161 cultures positive with both methods, the time of growth was shorter at least one week in 19 samples (12%) after using Präwozell N-BX and in 55 specimens (34%) homogenized by means of Ditalan E than in the parallel culture. The number of contaminations was 18 (0,85%) after Ditalan E pretreatment and 10 (0,47%) after Präwozell N-BX homogenization. Taking as a basis the directives concerning the cultural investigations for mycobacteria Ditalan E did not show better results than those found with Präwozell N-BX.