[Responsibilities of Weaning Centers during the COVID-19 Pandemic Outbreak - Recommendations for the Assignment of ICU Capacities in COVID-19 Patients as shown by the Berlin-Brandenburg POST-SAVE-Model]. / Aufgaben der Weaning-Zentren im Pandemiefall COVID-19.

The enormous increase in patients with severe respiratory distress due to the COVID-19 pandemic outbreak requires a systematic approach to optimize ventilated patient at risk flow. A standardised algorithm called "SAVE" was developed to distribute patients with COVID-19 respiratory distress syndrome requiring invasive ventilation. This program is established by now in Berlin. An instrumental bottleneck of this approach is the vacant slot assignment in the intensive care unit to guarantee constant patient flow. The transfer of the patients after acute care treatment is needed urgently to facilitate the weaning process. In a next step we developed a triage algorithm to identify patients at SAVE intensive care units with potential to wean and transfer to weaning institutions - we called POST SAVE. This manuscript highlights the algorithms including the use of a standardised digital evaluation tool, the use of trained navigators to facilitate the communication between SAVE intensive care units and weaning institutions and the establishment of a prospective data registry for patient assignment and reevaluation of the weaning potential in the future.

[Monitoring of None-invasive Ventilated Patients in a Home Care Setting]. / Prospektive Untersuchung der außerklinischen Versorgung und Therapiekontrolle bei nichtinvasiv heimbeatmeten Patienten.

There is a paucity of data about the at home monitoring and the outpatient setting and care of patients with non-invasive ventilation. We here show, in a prospective study, that both standardized outpatient care visits as well as quality of life monitoring at home are safe and feasible. Monitoring and managing the quality of care at home did not lead to an increase of non-elective hospitalisations or deterioration of respiratory disease burden.

[100 years DGP -100 years of pneumology in Germany]. / 100 Jahre DGP - 100 Jahre deutsche Pneumologie.

During the first half of its 100-year history, tuberculosis was predominant in the German Society of Pneumology (DGP). This led largely to the separation of pneumology from internal medicine, particularly in the universities. Since the 1960s, the spectrum of respiratory diseases has changed considerably. Asthma, COPD, lung cancer, and pneumonia today rank among the most widespread diseases. Numerous new diagnostic and therapeutic methods have induced dramatic changes in the field of pneumology. Today, pneumology, together with cardiology and gastroenterology, belongs to the major specialties of internal medicine. One of the most urgent tasks of the DGP is to improve the insufficient representation at German universities, and thus promote teaching and research in respiratory medicine.

[Diffuse abdominal pain in a 25-years old Indian female]. / 25-jährige indische Patientin mit diffusen abdominellen Beschwerden.

A 25-year-old female patient from India suffered from diffuse pain in the upper and lower abdomen, caused by infiltrative changes of the ovaries and intraabdominal fluid. The patient refused further examinations. 11 months later she endured additional pain of the thorax and the left shoulder. An opacity in the left lung and costal destruction was diagnosed. Puncture of the lung process showed a granulomatous inflammation including Langerhans cells. The puncture of a peritoneal thickening also showed inflammation. Microscopic examination was negative for mycobacteria, but PCR and the culture for Mycobacterium tuberculosis after 8 weeks were positive, with full sensibility, so treatment with RMP, INH, PZA and EMB was initiated. The lung process improved but the abdominal complaints were progressive due to a pyosyalpinx requiring surgical removal. The further course was uneventful. Two years after diagnosis the patient is in a good condition now.

Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (alpha and beta). The alpha subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of beta subunits in testis. The alpha and beta subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the alpha subunit is observed along the entire flagellum, whereas the short beta subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of alpha and beta subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

Multicentre evaluation of performance of C-reactive protein analysis over a one-year period.

This communication deals with a longitudinal evaluation of C-reactive protein (CRP) analysis during a one-year period using a single lot of liquid control sera (3 levels) (BIOREF-CRP levels 1, 2 and 3) in different laboratories. A total of 652 sets of data were returned from 20 participating laboratories using 13 different reagent-measuring device combinations. The use of the control materials was defined in a standard operating procedure. Data was returned to the organizers on a monthly basis and questions could be asked or problems presented during the evaluation period. Although the performance of different reagents varied, the control materials were shown to be stable over the whole of the evaluation period when stored at 4-7 degrees C in a refrigerator/cold room. Typical problems were encountered, examples of which are presented here in graphical and tabular form.

Evidence for extensive and non-specific translocation of oligopeptides across plasma membranes of mammalian cells.

After exposure of bovine aortic endothelial cells to various small peptides (tetra- to undeca-mer), extensive transport of the peptides across the plasma membrane was observed in the concentration range 10(-7) to 10(-2) M. The observed transport events, which contradict the generally anticipated poor permeability of peptides across plasma membranes, exhibited high complexity and showed no saturability up to a concentration of 10(-2) M. Evidence was found for the involvement of mdrp-like transporters as well as of energy-independent facilitated diffusion events. The peptide levels within the cells approximated those of the incubation solution within 30 min, indicating high capacity and velocity for the involved transport processes. Correspondingly, preloaded cells exported about 80% of the internalized peptide within 5 min at 37 degrees C. Analogous results were found after peptide exposure to several other mammalian cell types, indicating a more general importance of the transport phenomena described here. Our findings contradict the prevailing opinion that the often observed lack of activity of externally administered peptides against their targets within intact cells is accounted for primarily by poor cellular uptake and point to export processes counteracting the uptake to be more important in this context.

Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically.

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription--PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100-500 s at concentrations of substance P from 50 to 5 microM). Degranulation in response to intracellularly applied substance P was inhibited by GDPbetaS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein alpha subunits subsequent to their translocation across the plasma membrane.

Extensive cellular uptake into endothelial cells of an amphipathic beta-sheet forming peptide.

Extensive internalization into endothelial cells has been found for a water soluble amphipathic 26-mer beta-sheet peptide (FLUOS-DPKGDPKGVTVTVTVTVTGKGDPKPD-NH2; VT5). With the D-Val13,D-Thr14 di-D-amino acid analog of VT5 (DD-VT5), exhibiting an identical primary structure but no propensity to adopt a beta-sheet conformation, only about 5% of the cellular uptake of VT5 was found. The mechanism of entry of VT5 into the cells remained unclear, but proved to be energy, temperature and pH dependent and, therefore, clearly distinct from that reported for helical amphipathic peptides. No detectable cytotoxicity, high solubility in water and the found extensive entry into endothelial cells make VT5 appear a good lead for developing new types of vectors for delivering oligonucleotides and peptides into intact cells.

Folding and cell surface expression of the vasopressin V2 receptor: requirement of the intracellular C-terminus.

We characterized truncations of the human vasopressin V2 receptor to determine the role of the intracellular C-terminus (comprising about 44 amino acids) in receptor function and cell surface expression. In contrast to the wild-type receptor, the naturally occurring mutant R337X failed to confer specific [3H]AVP binding to transfected cells. In addition, no vasopressin-sensitive adenylyl cyclase was detectable in membrane preparations of these cells. Laser scanning microscopy revealed that c-myc epitope- or green fluorescent protein-tagged R337X mutant receptors were retained within the endoplasmic reticulum. Increasing the number of C-terminal residues (truncations after codons 348, 354 and 356) restored G protein coupling, but revealed a length-dependent reduction of cell surface expression. Replacement of positively charged residues within the C-terminus by glutamine residues also decreased cell surface expression. A chimeric V2 receptor with the C-terminus replaced by that of the beta2-adrenergic receptor did not bind [3H]AVP and was retained within the cell. These data suggest that residues in the N-terminal part of the C-terminus are necessary for correct folding and that C-terminal residues are important for efficient cell surface expression.

Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells.

We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.

Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation.

Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 +/- 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 +/- 0.17 nmol/mg of protein after reoxygenation vs control (0.41 +/- 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 +/- 0.01 to 0.11 +/- 0.02 nmol/mg of protein (P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E [(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)] were effective scavengers of .OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard (EC50: 12, 5 and 15 microM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 microM U83836E, and 5 microM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 +/- 7, 51 +/- 10 and 48 +/- 6%, respectively (P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.

p53 mutations and codon 213 polymorphism of p53 in lung cancers of former uranium miners.

PURPOSE: There is a high prevalence of G-->T transversions of p53 in lung cancers of smokers. One study has reported a special "hotspot" mutation at codon 249 of p53 in lung cancers of former uranium miners. The aim of our study was to look for mutational spectra of p53 in former German uranium miners with lung cancers. METHODS: We investigated 16 patients with lung cancer who had worked as uranium miners in Germany and 13 lung cancer patients without a mining history of the same region. By means of the polymerase chain reaction and sequencing we looked for mutations in exons 5 7 of the p53 gene. RESULTS: We could not find any suggestion of hotspot mutations. The only G-->T mutation in former uranium miners was detected in the only nonsmoker. In 3 patients (19% of the total) we found a codon 213/3 polymorphism. CONCLUSIONS: The results indicate that G-->T transversions do not seem to be very common mutations in p53 in lung cancers probably caused by radiation. Therefore, p53 may be mutated early in lung cancer development if radiation exposure is a critical factor in carcinogenesis. In accordance with studies of thyroid cancer patients in the Chernobyl region, our results may indicate an overrepresentation of codon 213/3 polymorphism in p53 in radiation-caused cancers.

Astrocytes induce manganese superoxide dismutase in brain capillary endothelial cells.

Astrocytes induce blood-brain barrier (BBB) properties in brain endothelial cells (EC)*O(2)*, generated in blood and EC, opens the BBB. Hence, high activity of superoxide dismutase (SOD) is a prerequisite for normal BBB function. Therefore, the influence of rat astrocytes on the expression of manganese (Mn)SOD in rat EC was investigated in two coculture models of the BBB, allowing either exchange of soluble factors or additionally cellular contacts. Activity, protein content and mRNA expression of endothelial MnSOD were significantly increased in both coculture models in comparison to monoculture by soluble astrocytic factors, such as cytokines. High activity of endothelial MnSOD may be considered as a further essential property of the BBB, which is induced and maintained by astrocytes.

Measurement of intracellular Ca2+ changes using novel caged cyclic nucleotides and confocal laser scanning microscopy.

The intention of this study is to explore the applicability of confocal microscopy in conjunction with the use of caged cyclic nucleotide derivatives. The methodological potential of UV laser confocal microscopy has been assessed. It is shown that illumination of a single cell or a small area of a single cell is possible, whereby the intracelluar Ca2+ signal is measured at illuminated and non-illuminated cells. Such measurements do not have a high time resolution because of the specific system parameters. However, with an N2 pulse laser (not part of the standard microscope set-up), Ca2+ signals with a time resolution of around 100 ms have been measured. This facilitates investigation of the kinetics of Ca2+ influx. Intracellular Ca2+ measurements at HEK293 and sperm cells have been made here. For sperm cells the advantages of confocal microscopy are best evidenced in conjunction with the use of caged cyclic nucleotides; a cyclic nucleotide-gated Ca2+ influx at the tail of these cells has thereby been demonstrated for the first time.

Synthesis, photochemistry and application of (7-methoxycoumarin-4-yl)methyl-caged 8-bromoadenosine cyclic 3',5'-monophosphate and 8-bromoguanosine cyclic 3',5'-monophosphate photolyzed in the nanosecond time region.

New caged derivatives of hydrolysis-resistant 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP) and 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP) are described. The compounds are the axial and equatorial isomers of the (7-methoxycoumarin-4-yl)methyl (MCM) esters of cyclic nucleotides. Synthesis is accomplished by treatment of 4-bromomethyl-7-methoxycoumarin with the tetra-n-butylammonium salts of the 8-bromo-substituted cyclic nucleotides or with the free acids of 8-Br-cAMP and 8-Br-cGMP in the presence of silver(I) oxide. MCM-caged 8-Br-cAMP and MCM-caged 8-Br-cGMP liberate 8-Br-cAMP and 8-Br-cGMP during irradiation with ultraviolet light within a few nanoseconds. They show favorable absorption properties and quantum yields and are resistant to hydrolysis in aqueous buffer solutions. The moderate fluorescence properties of the caged compounds in comparison with the strongly fluorescent 4-hydroxymethyl-7-methoxycoumarin (MCM-OH) photoproduct allow the indirect estimation of the amount of photolytically released cyclic nucleotides in aqueous buffer solutions using fluorescence measurements. Their usefulness for physiological studies has been examined in a mammalian cell line expressing the cyclic nucleotide-gated ion channel of bovine olfactory sensory neurons using the patch-clamp technique and confocal laser scanning microscopy. The caged compounds serve as efficient and rapid intracellular sources of 8-Br-cAMP and 8-Br-cGMP. However, at least in HEK 293 cells, fluorescence signals cannot be used to monitor the photolysis of MCM-caged 8-Br-cAMP and 8-Br-cGMP, due to quenching of the fluorescence of MCM-OH.

Hemodynamic response and effects on myocardial energetics of 3-cyano-2-morpholino-5-(pyrid-4-yl)pyridine (AWD 122-14) in anesthetized minipigs.

AWD 122-14, a new positive inotropic and vasodilating agent, was investigated in comparison to amrinone, milrinone and dopamine in anesthetized minipigs. AWD 122-14 (1.17.10(-7)-37.5.10(-7) mol/kg) increased dose-dependent left ventricular contractility (LV dp/dtmax) (122x5 +/- 11x3%; ED50 = 8.1x10(-7) to mol/kg). Dopamine (2.64x10(-8)-21x12 x 10(-8) mol/kg) in comparison increased contractility up to 153.1 +/- 44.9% of control value and is about 20 times more potent than AWD 122-14 at the ED50 value and about 10 times more potent at the ED30 value. Amrinone (1.60x10(-6)-16.90x10(-6) mol/kg) and milrinone (1.48x10(-7)-23.70x10(-7) mol/kg) only slightly increased contractility in anesthetized minipigs, but they appear to posses a similar pharmacological profile like AWD 122-14. The hemodynamic effects were associated with an increase in myocardial oxygen consumption (E1: 18.8 +/- 10.0%) due to the marked increases in LV dp/dtmax and heart rate. LVMW was unchanged and LVSW decreased (-29.0 +/- 10.2%) after application of AWD 122-14. The reduction in left ventricular work (LVMW, LVSW) and the increase in myocardial oxygen consumption led to a decrease of left ventricular external mechanical efficiency of the non-failing minipig heart (Etam: -21.1 +/- 9.4%). Additional hemodynamic effects of AWD 122-14 were studied under calcium channel blockade (verapamil, nifedipine). After pretreatment with verapamil the agent (1.17.10(-7)-18.75.10(-7) mol/kg i.v.) increased left ventricular contractility between 42.9 +/- 41.6% and 58.5 +/- 33.3%. After pretreatment with nifedipine the agent induced a dose-dependent increase in LV dp/dtmax between 11.1 +/- 7.7% and 47.8 +/- 23.7%.(ABSTRACT TRUNCATED AT 250 WORDS)