[Analgesia in the emergency medical service: comparison between tele-emergency physician and call back procedure with respect to application safety, effectiveness and tolerance]. / Analgesie im Rettungsdienst: Vergleich zwischen Telenotarzt- und Callback-Verfahren hinsichtlich Anwendungssicherheit, Wirksamkeit und Verträglichkeit.

BACKGROUND: Acute pain is a common reason for calling emergency medical services (EMS) and can require medication depending on the pain intensity. German EMS personnel feel strong pressure to reduce a patient's pain but are restricted by law. Currently, German federal law only allows the administration of opioid-containing drugs by or on the order of a physician, while in other European countries (e.g. Switzerland and The Netherlands) the administration of opioid-based analgesia by trained and certified paramedics is common practice. Consequently, a patient in Germany experiencing acute pain needs the attendance of an emergency physician in EMS missions. According to international standards pain reduction on the numeric rating scale (NRS) score by ≥2 or a NRS score ≤4 at the end of the patient transport is considered to be adequate. OBJECTIVE: Comparison of two different algorithm-based concepts for analgesia with consultation of a physician analyzing the efficacy, tolerance and safety of application. MATERIAL AND METHODS: In a retrospective cohort study in two different regions, two physician-supported algorithm-based analgesia concepts, a call back-supported concept (EMS Schleswig-Holstein: RKiSH) and a tele-EMS physician-based concept (EMS Aachen: RDAC), were compared over 2 years. The call back-supported concept is based on specific algorithms and certification of EMS personnel. In Aachen, the tele-EMS physician is integrated into the routine EMS system and includes immediate vital data transmission. RESULTS: Over a period of 2 years call back-supported analgesia was administered in 878 cases (2016: 428, 2017: 450) and telemedically assisted analgesia was used in 728 cases (2015: 226, 2016: 502). Call back vs. telemedicine: initial NRS scores were 9 (8-10) and 8 (6-9), respectively (p < 0.0001); NRS scores were reduced by 4 (3-5) and 5 (3-6), respectively (p = 0.0002), leading to mean NRS scores of 4 (3-6) vs. 3 (2-4), respectively (p < 0.0001) at patient handover/emergency room arrival. Clinically relevant pain reduction was achieved in both groups. Complete NRS documentation was conducted in 753 (85.8%) vs. 673 (92.4%) cases, respectively, p = 0. Severe adverse events did not occur in either of the groups. CONCLUSION: The administration of analgesia by EMS personnel with teleconsultation of a physician is effective and has a low rate of complications, particularly morphine. Overall, algorithm-based call back-supported as well as telemedically supported analgesia concepts based on regular training improve the management of pain in the prehospital setting. In addition, the resources of the emergency physician remain available for life-threatening emergencies. The training, certification and supervision of EMS personnel is very important in both systems to ensure the best pain management care and patient safety. Adjustments to the federal law on the administration of analgesics would facilitate the realization of algorithm-based concepts by paramedics as pain reduction could be performed with delegation by a medical director without consulting another physician.

A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

Investigation of prevalence and regulation of expression of progression associated protein (PAP).

Progression Associated Protein (PAP), a new transmembrane receptor encoded by 157 amino acids was identified as a progression marker by differential display techniques of mammary carcinoma cell lines with different metastatic properties. PAP has been isolated independently by several groups in humans, mouse and rabbit, and has received different designations (TMP, EMP-1, CL-20 and B4B). As a first step towards understanding the biological function of PAP we have investigated prevalence and regulation of expression of PAP in human tissues, primary cells and tumor cell lines. PAP is expressed in most, but not all human tissues and investigation of several human mammary carcinoma cell lines with different metastatic characteristics revealed a correlation between expression of PAP and their invasive and metastatic properties. Expression of PAP under proliferation and growth-arrest conditions was investigated by a combination of Northern blot and FACS analysis. Expression of PAP was associated with proliferative status of the cells and its down-regulation was linked to induction of G1 arrest. These data indicate that PAP is inversely regulated compared to PMP22, a growth-arrest gene which belongs to the same gene family as PAP.

New genes potentially involved in breast cancer metastasis.

Identification of new genes involved in the pathogenesis of breast cancer opens new avenues for improved diagnostic markers and new molecular targets for improved treatment of this malignancy. In the following we review genes with proved involvement in invasion and metastasis of breast cancer as well as genes which exhibit an expression pattern that correlates with invasion and metastasis.

Differential gene expression in mammary carcinoma cell lines: identification of DRIM, a new gene down-regulated in metastasis.

Differential display technique was applied to a pair of cell lines derived from human breast carcinoma cell line MDA-MB 435 with metastatic and non-metastatic properties in the nude mouse system, with the objective to isolate genes involved in metastasis. DRIM (Down-Regulated In Metastasis) was the only gene found to be differentially expressed in this system. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C. elegans. The protein contains a conserved positively charged tail and several HEAT repeats, designated after four functionally characterized proteins in which the repeat was detected. Most of the hydrophobic regions of DRIM can be assigned to HEAT repeats. Expression of DRIM at the RNA level was investigated in several normal tissues and tumor cell lines.

Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA.

DNA of two yeast artificial chromosomes (YACs) containing selectable human genes was transferred by microinjection to rodent cells in tissue culture. The human hypoxanthine phosphoribosyltransferase (HPRT) gene, spanning 45 kb, is contained on the 660-kb YAC yHPRT as described elsewhere. The human phosphoribosylglycinamide formyltransferase (GART) gene, spanning approximately 40 kb, is contained on the 590-kb YAC yGART2 as described previously. YAC DNA was isolated from pulsed-field gels and microinjected into mammalian cells in which the human HPRT and GART genes can be selected. The cell lines that were selected contain the entire human genes. Some of the cell lines contain multiple copies of the genes integrated at the same chromosomal position. The YAC yGART2 could not be purified away from natural yeast chromosomes of similar size, and the cell lines into which the human GART gene was introduced contain variable amounts of yeast DNA in addition to the human DNA.

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.

tRNA binding sites on the subunits of Escherichia coli ribosomes.

Programmed 30 S subunits expose only one binding site, to which the different classes of tRNA (deacylated tRNAPhe, Phe-tRNAPhe, and N-acetylphenylalanyl (AcPhe)-tRNAPhe) bind with about the same affinity. Elongation factor Tu within the ternary complex does not contribute to the binding of Phe-tRNA. Binding of acylated or deacylated tRNA to 30 S depends on the cognate codon; nonprogrammed 30 S subunits do not bind tRNA to any significant extent. The existence of only one binding site/30 S subunit (and not, for example, two sites in 50% of the subunits) could be shown with Phe-tRNAPhe as well as deacylated tRNAPhe pursuing different strategies. Upon 50 S association the 30 S-bound tRNA appears in the P site (except the ternary complex which is found at the A site). Inhibition experiments with tetracycline demonstrated that the 30 S inhibition pattern is identical to that of the P site but differs from that of the A site of 70 S ribosomes. In contrast to 30 S subunits the 50 S subunit exclusively binds up to 0.2 and 0.4 molecules of deacylated tRNAPhe/50 S subunit in the absence and presence of poly(U), respectively, but neither Phe-tRNA nor AcPhe-tRNA. Noncognate poly(A) did not stimulate the binding indicating codon-anticodon interaction at the 50 S site. The exclusive binding of deacylated tRNA and its dependence on the presence of cognate mRNA is reminiscent of the characteristics of the E site on 70 S ribosomes. 30 and 50 S subunits in one test tube expose one binding site more than the sum of binding capacities of the individual subunits. The results suggest that the small subunit contains the prospective P site and the large subunit the prospective E site, thus implying that the A site is generated upon 30 S-50 S association.

Large-scale synthesis of the mRNA analogue C17AUGA4C17.

The large-scale synthesis of the mRNA analogue C17AUGA4C17 is described. This molecule is 41 bases in length and contains three unique codons in its central region. tRNA binding studies at 6 mM Mg2+ in the absence and presence of the labeled heteropolymer show the mutual stimulation of tRNA and mRNA binding and demonstrate that this mRNA provides a simple and suitable system for the analysis of the reactions of the ribosomal elongation cycle.

tRNA binding capacities of ribosomal subunits from the archaebacterium Halobacterium halobium.

tRNA saturation experiments were performed with ribosomal subunits from the extreme halophilic archaebacterium Halobacterium halobium. In the presence of poly(U) the 30S subunit could bind equally well one AcPhe-tRNAPhe, Phe-tRNAPhe, or deacylated tRNAPhe molecule, respectively. Binding experiments with a mixture of two differently labeled tRNA species revealed that all three kinds of tRNA bound to one and the same binding site on the 30S subunit. Poly(U) dependent binding to the 50S subunit was insignificant for AcPhe-tRNA and Phe-tRNA. In the absence of poly(U) both AcPhe-tRNAPhe and Phe-tRNAPhe showed no significant binding to either subunit, whereas the binding of deacylated tRNAPhe could not be clearly determined. These results are in good agreement with those obtained from ribosomal subunits of the eubacterium Escherichia coli.

Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells.

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.

Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

[Recent developments in obstetric regional anesthesia. A review of experiences at the Eppendorf University Hospital]. / Aktuelle Entwicklungen der geburtshilflichen Regionalanästhesie. Eine Ubersicht am Beispiel des Universitäts-Krankenhauses Eppendorf.

OBJECTIVES: The study investigates changes of anaesthesia practice in obstetric patients of a University Hospital over a six years' period. METHODS: Between 1993 and 1998 data of 7476 deliveries were collected by the perinatal documentation system of the Obstetric Department and by computerized anaesthesia protocols of the Department of Anesthesiology. Since combined spinal-epidural anaesthesia with sufentanil was introduced in 1997, all patients with subarachnoid techniques were prospectively examined between 1997 and 1998. RESULTS: While the total number of deliveries decreased over the years, the number of patients undergoing anaesthetic treatment increased continuously. In parallel, the number of patients with regional anaesthesia increased between 1993 and 1998 from 14.3% to 34.8%. The cesarean delivery rate increased from 23.7% to 28.7% with an increasing number of patients receiving regional anaesthesia for cesarean section (1993: 25.3% vs. 1998: 62.1%). The number of emergency cesarean deliveries performed in regional anaesthesia increased to 19.3% in 1998. The number of neonates with an umbilical artery pH below 7.2 decreased from 18% in 1993 to 11% in 1998. The success rate of regional anaesthesia increased from 88.2% in 1993 to 97.5% in 1998. Combined spinal-epidural anaesthesia provided greater pain reduction when compared with epidural anesthesia (VAS -81 +/- 12 vs. -68 +/- 18). Early vasopressor administration resulted in a decrease of hypotension from 40% to 14% in spinal and from 21% to 13% in combined spinal-epidural anaesthesia. The incidence of postdural puncture headache after subarachnoid anaesthesia was 2.4%. DISCUSSION: Epidural and subarachnoid application of sufentanil appears to enhance the success rate of obstetric regional anaesthesia. Subarachnoid techniques such as spinal or combined spinal-epidural anaesthesia showed a high effectiveness, low incidence of side effects and high degree of patients' satisfaction.

Physical calibration of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease (RARE) cleavage.

Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.

Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities.

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.

The allosteric three-site model for the ribosomal elongation cycle. Analysis with a heteropolymeric mRNA.

Ribosomal tRNA binding studies and functional tests were performed at 6 mM Mg2+ using the mRNA analogue C17AUGA4C17 which contains three unique codons in its central region. The following results were obtained. 1) The relative binding affinities of 20 different deacylated tRNAs to nonprogrammed 70 S ribosomes were assessed and were found to vary substantially. 2) When added as the first tRNA, fMet-tRNA and deacylated tRNAs (but not N-acetylated aminoacyl-tRNAs) can bind to internal codons of the mRNA and are therefore suitable for setting the reading frame via codon-anticodon interaction in the peptidyl-tRNA site (P site). 3) After prefilling the P site with deacylated tRNA, the exit site for deacylated tRNA (E site) can be quantitatively occupied only if the cognate codon is present at that site. 4) The translocation of peptidyl-tRNA from the aminoacyl-tRNA site (A site) to the P site is not accompanied by a release of deacylated tRNA. The codon sequence excludes a release and rebinding of deacylated tRNA to the newly exposed A site. Rather, the deacylated tRNA is cotranslocated from the P to the E site where it remains stably bound. 5) After one round of elongation, addition of an A site ligand triggers the dissociation of deacylated tRNA from the E site. Conversely, E site occupation reduces the affinity of the A site for N-acetylated aminoacyl-tRNA. Thus, A and E sites are allosterically linked via negative cooperativity. The results support the allosteric three-site model as an appropriate description of the ribosomal elongation cycle.