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Biomedical subjects

U Pachmann

Publications and source records attributed to U Pachmann.

At least 19 recordsLinked to original sources
PubMed1999

First workshop for detection of heparin-induced antibodies: validation of the heparin-induced platelet-activation test (HIPA) in comparison with a PF4/heparin ELISA.

BACKGROUND: No data exist regarding the inter-laboratory reproducibility of the heparin-induced-platelet-activation (HIPA) test, the most widely used functional assay in Germany for the detection of heparin-induced thrombocytopenia (HIT) antibodies. METHODS: Nine laboratories used an identical protocol to test eight different sera with the HIPA test. Five laboratories also tested the sera with a platelet factor 4 (PF4)/heparin-complex ELISA. Cross-reactivity with danaparoid-sodium was assessed using 0.2 aFXa units instead of heparin in the HIPA test. RESULTS: Two of nine laboratories had no discrepant HIPA test results. Four laboratories differed in one sample, one reported two discrepant results, and two laboratories reported more than two discrepant results. Cross-reactivity with danaparoid-sodium test results differed among laboratories. PF4/heparin ELISA results were identical in all five laboratories. CONCLUSION: The HIPA test requires strict quality control measures. Using both a sensitive functional assay (HIPA test) and a PF4/heparin ELISA will allow detection of antibodies directed to antigens other than PF4/heparin complexes as well as detection of IgM and IgA antibodies with PF4/heparin specificity.

Antibodies↗
PubMed1994

[Regular platelet donors--no risk to the donor].

It is not known whether frequent platelet donations provoke the platelet precount of the donor to rise or to decline. Therefore, with regard to the maximally possible safety of our donors we looked back onto the platelet counts of platelet donors who had given their platelets at constant 2-week intervals. Even frequent plateletphereses at 2-week intervals do not provoke thrombocytosis or thrombopenia for donors with normal platelet counts (> 200/nl, < 400/nl). Moreover, our donors appeared to regulate their platelet counts in an 'intelligent' manner.

Blood Donors↗
PubMed1990

Preparation of white cell-depleted blood. Comparison of two bedside filter systems.

Numerous techniques have been established for preparing white cell-poor blood, such as centrifugation, sedimentation, freezing, and filtration. All of these methods have disadvantages that restrict their practical use: they are time-consuming, they require the facilities of a blood bank, and the white cell-poor units cannot be stored. Therefore tests have been performed on two filter systems that make possible the depletion of white cells directly at the bedside. Both filters have a high white cell-removal rate. The number of residual white cells in 1 unit of packed red cells was calculated as 1.08 +/- 0.53 x 10(7) in one system and 1.54 +/- 0.71 x 10(7) in the other. The filters do not affect the filtered red cells. Pressure filtration is performed without significant loss of efficiency. One of the systems can filter 2 units via one filter; however, handling the filters is tedious, and both filters are characterized by a low red cell recovery. Despite these disadvantages, the tested filter systems provide an effective device for preparing white cell-poor blood at the bedside. They are a suitable alternative to the conventional methods.

Blood Platelets↗
PubMed1988

[Antibody detection in emergency transfusions. A comparison of 3 different methods].

We compared the manual Polybrene technique to three standard methods (albumin/Coombs, LISS/Coombs, and enzyme/papain) on 113 red cell antibodies. Polybrene identified 8 antibodies of the Rhesus system missed by standard methods, whereas 3 antibodies could only be detected by the standard techniques. Four antibodies were identified only in saline solution at room temperature; 6 were found by use of Polybrene exclusively in the additional Coombs phase. In addition, 61 antibodies were tested by 4 different LISS. No considerable differences in the quality of the various LISS were seen. The manual Polybrene test appears to be suitable for crossmatching and rapid antibody identification in emergency situations.

Blood Group Antigens↗
PubMed1985

Allocation of the suppressive activity of normal peripheral blood lymphocytes induced by diffusion chamber culture and Con A stimulation to the G2 phase of the cell cycle.

Peripheral blood mononuclear cells stimulated either by diffusion chamber culture or by a high Con A concentration exhibit suppressive activity under conditions where no increase in cell number takes place. Instead an accumulation of large cells is observed which, according to their DNA contents, are classified as cells in the G2 phase of the cell cycle. By elutriation separation the suppressive activity is shown to be confined to this cell cycle phase.

Adult↗
PubMed1983

Identification of the messenger (m) RNA coding for the constant fragment (c mu) of the heavy chain with cloned DNA in single cells by in situ hybridization.

Cells from a normal donor, a B-CLL and a T-ALL were labeled with an FITC conjugated rabbit antihuman IgM and hybridized with a rhodamine-conjugated cloned DNA coding for the c mu segment. The cells were measured simultaneously by immunofluorimetry for their surface content of IgM and their amount of m-RNA hybridizing with the cloned DNA. Thus it was possible to compare the transcription of the gene coding for the immunoglobulin mu chain and the expression of IgM in individual cells. It could be shown that besides the immunoglobulin mu containing B-cells, which were expected to contain the respective m-RNA, cells were found which did not express IgM but which were still positive for the m-RNA.

B-Lymphocytes↗
PubMed1981

Conformational states of yeast tRNA Phe in the complex with cognate and non cognate synthetases.

The influence of phenylalanyl-tRNA synthetase and seryl-tRNA synthetase on the conformation and structural kinetics of yeast tRNA Phe was investigated. Ethidium substituted for dihydrouracil at position 16 or 17 was used as a structural probe, showing the existence of three conformational states in tRNA. The distribution of states (T1, T2, T3) is changed only by the cognate synthetase towards T3 which probably is related to the X-ray structure. The binding of phenylalanyl-tRNA synthetase leads to an about 10-fold increase in the fast transition T1 in equilibrium or formed from T2 which has been assigned to changes in the anticodon loop conformation and to a 2-3 fold increase in the slow transition which probably extends to other parts of the tRNA molecule. The observed rates for the transition T2 in equilibrium or formed from T3 are close to that observed for the transfer of the activated phenylalanine to tRNA Phe. This raises the possibility that the conformational transition in tRNA is the rate limiting step in the charging reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1980

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation.

Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.

Adenosine Triphosphate↗
PubMed1978

Yeast seryl tRNA synthetase: interactions between the ATP binding site and the sites for tRNASer and L-serine.

T1 ribonuclease digestion of yeast tRNASer in the presence of seryl tRNA synthetase was used for monitoring the relationship between the substrate binding sites on the synthetase. It was found that (a) ATP displaces the tRNA from the synthetase with an effector affinity constant corresponding to the Km for ATP of 10 micron; (b) AMP and a number of nucleoside triphosphates, while influencing the rate of aminoacylation, do not displace the tRNA from the enzyme; (c) ADP and PPi inhibit the aminoacylation and the binding of tRNASer; (d) adenylyl diphosphonate is bound to the synthetase and lowers the protection of the tRNA against the nuclease attack in a similar way as does ATP; (e) interactions between the sites of L-serine and tRNASer could only be shown when both sites for serine were saturated and, in addition, the ATP analog or ADP was present. It is concluded that in seryl tRNA synthetase binding sites for ATP interact with the ones for tRNA as well as with the ones for serine. These findings contribute to the understanding of the mechanism of aminoacylation.

Adenosine Triphosphate↗
PubMed1976

On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition.

By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recognition process involving a first unspecific step where all tRNAs, cognate and non-cognate, are bound to the synthetase (scanning step). The identification of the cognate tRNA is then performed at the recognition site by a conformational transition of the tRNA . synthetase complex (identification step).

Amino Acyl-tRNA Synthetases↗