Recombination dynamics of type-II excitons in (Ga,In)As/GaAs/Ga(As,Sb) heterostructures.

(Ga,In)As/GaAs/Ga(As,Sb) multi-quantum well heterostructures have been investigated using continuous wave and time-resolved photoluminescence spectroscopy at various temperatures. A complex interplay was observed between the excitonic type-II transitions with electrons in the (Ga,In)As well and holes in the Ga(As,Sb) well and the type-I excitons in the (Ga,In)As and Ga(As,Sb) wells. The type-II luminescence exhibits a strongly non-exponential temporal behavior below a critical temperature of T c = 70 K. The transients were analyzed in the framework of a rate-equation model. It was found that the exciton relaxation and hopping in the localized states of the disordered ternary Ga(As,Sb) are the decisive processes to describe the dynamics of the type-II excitons correctly.

Inclusion of HPV testing in routine cervical cancer screening for women above 29 years in Germany: results for 8466 patients.

In a prospective cohort study 8466 women attending routine cervical cancer screening were recruited. Colposcopy was performed on women with any degree of atypia on cytology and/or a positive high-risk human papillomavirus (HPV)-DNA test (HC2; Hybrid Capture 2((c))), and for a randomly selected sample of 3.4% women with negative findings on both. Quality control included reviews of cytology, histology, colposcopy images and retesting of samples with polymerase chain reaction. Test diagnostic performances were based on 7908 women who had complete baseline and follow-up results. Routine histology identified 86 women with high-grade cervical intraepithelial neoplasia (CIN2+), which was confirmed by review histology in only 46 cases. Sensitivity of routine cytology for the detection of CIN2+ was 43.5%, with a specificity, positive predictive value (PPV), negative predictive value (NPV) of 98.0, 11.4 and 99.7%, respectively. Sensitivity of the HC2 test for the detection of CIN2+ was 97.8%, with a specificity, PPV and NPV, of 95.3, 10.9 and 100%, respectively. No high-grade neoplasia was detected in the randomly selected control group. A negative HPV-test result, even in combination with a positive Papanicolaou (Pap) result, virtually excluded any risk of underlying high-grade disease, but this was not the case for a negative Pap result. These data show that HPV testing is of value for the detection or exclusion of prevalent CIN in a routine cervical cancer-screening setting and could be used for further risk classification of women for follow-up management.

[Heart rate variability and chemoreflex sensitivity. Proved and new methods in risk prediction of malignant arrhythmias]. / Herzfrequenzvariabilität und Chemoreflexsensitivität. Bewährtes und Neues in der Risikoprädiktion maligner Herzrhythmusstörungen.

For the analysis of a disturbed autonomic function as a risk predictor for ventricular tachyarrhythmias, tonic and phasic procedures are available. The heart rate variability as a tonic procedure shows significant differences between patients with an increased risk of malignant arrhythmias and patients without increased risk. This can be demonstrated in patients with survived myocardial infarction, dilated cardiomyopathy and congestive heart failure. But the positive predictive value amounts only to about 50%. The chemoreflex sensitivity as a new phasic method represents a new possibility for the evaluation of a dysfunction of autonomic reflex arches. It is reduced due to a decreased left ventricular function and increasing age. Furthermore, it shows significant differences between patients with ventricular arrhythmias and patients without. The predictive accuracy concerning malignant ventricular arrhythmias in a population of 60 patients in the chronic postinfarction stadium amounts to 55%, the relative risk to 7.6. Thus, this method shows a high predictive power, but more investigations in larger patient cohorts are necessary to corroborate these results.

Identification of the binding site for the extrahelical target base in N6-adenine DNA methyltransferases by photo-cross-linking with duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position.

DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5'-TCGA-3' DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50-60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M. CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M. TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases.

Functional roles of the conserved aromatic amino acid residues at position 108 (motif IV) and position 196 (motif VIII) in base flipping and catalysis by the N6-adenine DNA methyltransferase from Thermus aquaticus.

The DNA methyltransferase (Mtase) from Thermus aquaticus (M.TaqI) catalyzes the transfer of the activated methyl group of S-adenosyl-L-methionine to the N6 position of adenine within the double-stranded DNA sequence 5'-TCGA-3'. To achieve catalysis M.TaqI flips the target adenine out of the DNA helix. On the basis of the three-dimensional structure of M.TaqI in complex with the cofactor and its structural homology to the C5-cytosine DNA Mtase from Haemophilus haemolyticus, Tyr 108 and Phe 196 were suggested to interact with the extrahelical adenine. The functional roles of these two aromatic amino acid residues in M.TaqI were investigated by mutational analysis. The obtained mutant Mtases were analyzed in an improved kinetic assay, and their ability to flip the target base was studied in a fluorescence-based assay using a duplex oligodeoxynucleotide containing the fluorescent base analogue 2-aminopurine at the target position. While the mutant Mtases containing the aromatic amino acid Trp at position 108 or 196 (Y108W and F196W) showed almost wild-type catalytic activity, the mutant Mtases with the nonaromatic amino acid Ala (Y108A and F196A) had a strongly reduced catalytic constant. Y108A was still able to flip the target base, whereas F196A was strongly impaired in base flipping. These results indicate that Phe 196 is important for stabilizing the extrahelical target adenine and suggest that Tyr 108 is involved in placing the extrahelical target base in an optimal position for methyl group transfer. Since both aromatic amino acids belong to the conserved motifs IV and XIII found in N6-adenine and N4-cytosine DNA Mtases as well as in N6-adenine RNA Mtases, a similar function of aromatic amino acid residues within these motifs is expected for the different Mtases.

[Assessment of the anti-ischemic effect in patients with therapy refractory angina pectoris in end-stage coronary heart disease--results of chronic intermittent urokinase therapy]. / Antiischämischer Wirkungsnachweis bei Patienten mit therapierefraktärer Angina pectoris im Endstadium der koronaren Herzkrankheit--Ergebnisse zur chronisch-intermittierenden Urokinasetherapie.

Patients with refractory angina pectoris and end-stage coronary artery disease represent an increasing clinical problem. Numbers of these patients will increase in the future for improved survival due to effective secondary prevention of coronary artery disease. Next to the evaluation of clinical symptoms non-invasive objective parameters of myocardial ischemia are of major relevance before initiation of alternative treatment modalities and for verification of antiischemic effectiveness. Based on our own experience it can be shown that in these patients testing which is mainly based on the patients physical exercise capacity is only of limited value due to the early occurrence of clinical symptoms. Furthermore diffuse perfusion abnormalities reduce the sensitivity of electrocardiographic and scintigraphic detection of ischemic changes. In contrast indirect measures of ischemia relating to the systolic or diastolic function of the left ventricle like doppler-echocardiography and radionuclide ventriculography seem to be promising approaches. This is confirmed by the results from the application of long-term intermittent urokinase therapy. Long-term intermittent urokinase therapy leads to an absolute enhancement of myocardial perfusion, which makes this approach superior to other medical interventions which are mainly based on a reduction of cardiac work-load.

Moderate discrimination of REP-1 between Rab7 x GDP and Rab7 x GTP arises from a difference of an order of magnitude in dissociation rates.

The kinetics of the interaction of Rab7 with REP-1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP-1 has higher affinity for the GDP bound form of Rab7 (Kd=1 nM) than for the GTP bound form (Kd=20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast (k+1 = ca. 10[7] M[-1] s[-1]) and concentration dependent while the second represents a slow equilibration (k+2 + k-2 = 3.5 s[-1]) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s[-1] for Rab7 x GDP and 0.2 s[-1] for Rab7 x GTP).

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.

[Chemoreflex and baroreflex sensitivity in patients with survived sudden cardiac death]. / Chemoreflex- und Baroreflexsensitivität bei Patienten mit überlebtem plötzlichem Herztod.

INTRODUCTION: To evaluate patients with an increased risk of sudden cardiac death the analysis of ventricular late potentials, heart rate variability and baroreflexsensitivity is helpful. However, the prediction of malignant arrhythmic events cannot be performed with sufficient accuracy. For a better identification of high risk patients other methods are necessary. In this study the impact of the chemoreflexsensitivity for the prediction of ventricular tachyarrhythmias was investigated. METHODS: Out of 44 patients included in the study, 23 were survivors of sudden cardiac death (SCD). Seven patients suffered from sustained monomorphic ventricular tachycardias, 14 had no arrhythmic events in their prior history. For the investigation of the baroreflexsensitivity (BRS) systolic blood pressure was augmented by Norfenefrin (Novadral) and the resulting increase of RR-intervals was measured in the surface-ECG. For determination of the chemoreflexsensitivity (ChRS) the ratio of the RR-interval-shift and the blood pressure shift during a 5-min inhalation of oxygen with a nose mask was formed. RESULTS: Patients with aborted SCD showed significantly decreased values for the ChRS compared to those patients without an arrhythmic event in their prior history (2.49 +/- 1.86 vs. 6.75 +/- 6.79 mm Hg, p < 0.001). In contrast, for the BRS no significant differences could be found (5.23 +/- 3.95 vs. 5.34 +/- 3.10 mm Hg, p = n.s.). Patients with aborted sudden cardiac death and inducible tachyarrhythmias during the electrophysiologic study showed significantly lower values of BRS and ChRS compared to patients without inducibility. CONCLUSION: As a new method for identification of patients with an increased risk of sudden cardiac death the analysis of chemoreflexsensitivity seems feasible and indicates an increased arrhythmic risk with a high sensitivity. The predictive impact has to be corroborated in larger patient collectives by prospect studies.

Construction of a deletion library using a mixture of 5'-truncated primers for inverse PCR (IPCR).

A quick in vitro mutagenesis method for the construction of nested deletion libraries was developed. Many deletions can be obtained in a single inverse PCR (IPCR) by replacing one of the two primers with a mixture of 5'-truncated oligodeoxynucleotides. Since chemical DNA synthesis proceeds from the 3'to the 5'end, such a mixture of 5'-truncated oligodeoxynucleotides can easily be obtained in a single automated DNA synthesis under reduced coupling efficiency. This deletion mutagenesis method yields many different deletions in a defined short DNA segment and is, therefore, best suited for a deletion analysis at base pair level. Applications might include functional analysis of regulatory DNA segments and protein engineering work that requires libraries for the expression of N-terminal, C-terminal or internal truncated proteins as well as fusion proteins having different splice sites.

Pharmacokinetics of denbufylline in healthy elderly subjects.

A single 100 mg oral dose of denbufylline was administered in an open study to ten healthy, elderly subjects in order to characterise the pharmacokinetics of parent compound and its major pharmacologically active, circulating metabolites. Plasma concentrations were determined using a combination of GC and HPLC methods. Since denbufylline plasma concentrations were close to the assay limit of reliable determination, the disposition of the compound was assessed in terms of the pharmacokinetic parameters of three active metabolites. The general pharmacokinetic behaviour of debufylline was similar to that observed in young, healthy subjects, although plasma concentrations of the metabolites were higher and half-lives tended to be longer in the elderly group. None of the subjects presented any adverse events during the study.