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P Thamm

Publications and source records attributed to P Thamm.

At least 19 recordsLinked to original sources

Pain therapy following joint replacement.A randomized study of patient-controlled analgesia versus conventional pain therapy.

A prospective randomized trial in 42 patients undergoing elective total hip or knee arthroplasty under general anaesthesia was carried out to evaluate the efficacy of patient-controlled analgesia (PCA) versus demanded conventional pain therapy (CPT) for controlling postoperative pain. Four patients had to be excluded from the study (2 postoperative confusion, 1 elevated piritramid dosage caused by chronic pain therapy, 1 stressed by PCA pump handling). PCA group (n = 19) received piritramid via PCA pump, CPT group (n = 19) received tramadol (oral or intramuscularly) or piritramid intravenously. PCA or CPT was started in the intensive care unit. Pain was measured with a standard 100 mm visual analogue scale (VAS) for 60 h postoperatively. Over this period of time, no significant differences were found in the pain score of both groups, nor did the incidence of side-effects differ significantly. The PCA group required on average twice as much piritramid-equivalent than the CPT group (P < 0.001). Patient satisfaction was good in both groups, but significantly better in the PCA group (P < 0.01), although the measured postoperative individual pain scores were above the preoperatively determined individual subjective pain threshold in the majority of both groups. From these results we draw the conclusion that even if the patients feel satisfied by the pain therapy administered, the majority are objectively treated below their individual subjective pain threshold.

Administration, Oral↗

Insulin receptors on cultured hypothalamic cells: functional and structural differences from receptors on peripheral target cells.

We have studied the functional and structural characteristics of insulin receptors on cultured rat hypothalamic cells. The receptors on these cells are specific for insulin, but have a lower binding affinity than that measured in nonneuronal tissues. Neither acute (2-h) nor long term (24-h) exposure of the hypothalamic cells to high insulin concentrations resulted in receptor down-regulation. However, insulin is internalized in these cells and accumulated in the presence of the lysomotropic agent chloroquine. Acute exposure to insulin does not alter initial rate of 2-deoxyglucose transport in hypothalamic cells, but does cause a stimulation of aminoisobutyric acid uptake. Photoaffinity labeling of the receptors of the hypothalamic cells with a biologically active photosensitive insulin revealed a major specifically labeled band of 115K mol wt and a minor band of 40K mol wt under disulfide-reducing conditions compared to bands of 125K and 90K mol wt seen after labeling of the insulin receptors of adipocytes. The receptor proteins in hypothalamic cells under nonreducing conditions (420K, 370K, and 310K mol wt) were also smaller than those in adipocytes. Thus, the insulin receptors of cultured hypothalamic cells differ from insulin receptors on peripheral target tissues in both functional and structural aspects.

Adipose Tissue↗

Synthesis of the porcine intestinal peptide PHI and its 24-glutamine analogue.

PHI--a new candidate hormone from porcine intestinal tract-- corresponds to a linear heptacosapeptide amide of remarkable sequence homology to the known members of the glucagon family, particularly to the vasoactive intestinal peptide (VIP) and secretin. The position 24 usually occupied by an aminodicarboxylic acid omega-amide, in the present case, however, carries a glutamic acid, thus opening the question of whether this structural feature is related to desamidation in one of the isolation and characterization steps or of whether it is significant for this peptide factor. Consequently the heptacosapeptide amides corresponding to the proposed primary structure and to its 24-glutamine analogue have been synthesized. Comparative chromatographic and biological studies on the natural and the two synthetic products have confirmed the correctness of the primary structure proposed for the isolated PHI. Since [24-glutamic acid] and [24-glutamine]PHI exhibit no significant differences in their biological potencies, the main question is still open of whether the position 24 in native PHI is occupied by the aminodicarboxylic acid omega-amide (glutamine) or by N-substituted derivatives (N-glycosyl).

Amino Acid Sequence↗

[Syntheses of human little gastrin-I and its leucine-15, norleucine-15 and methoxinine-15 analogs].

Human little gastrin-I is known to exhibit a high tendency to air-oxidation of its methionine-15 residue to the corresponding S-oxide derivative, with concomitant loss of biological activity. Since its leucine-15 analog, even if fully biologically active, differs significantly from the parent hormone in the immunological properties, the norleucine-15 and methoxinine-15 analogues were synthetized. For the required comparative analyses new syntheses of human little gastrin-I and of its leucine-15 analog were additionally elaborated. Upon an optimized condensation of the fragments, followed by the deprotection step, partition chromatography as well as preparative high-performance liquid chromatography led to the desired gastrins in satisfactory yields and high degree of purity as judged by the expected and known side products.

Amino Acid Sequence↗

Internalization and molecular processing of insulin receptors in isolated rat adipocytes.

The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with 125I-labeled NAPA-DP-insulin were identified by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands of Mr 330,000, 295,000, and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16 degrees C were immediately trypsinized, all of the receptor bands were degraded into lower molecular weight fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 37 degrees C for 1 hr prior to trypsin exposure, approximately equal to 30% of the receptors were found to be trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37 degrees C (but not at 16 degrees C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16 degrees C and during the subsequent incubation at 37 degrees C showed that this agent (i) increased the trypsin-insensitive (intracellular) receptor pool, (ii) blocked conversion of the Mr 125,000 subunit into the Mr 115,000 component, and (iii) prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulin-receptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).

Adipose Tissue↗

Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.

We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.

Affinity Labels↗

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.

Animals↗

[Cholecystokinin-pancreozymin synthesis. Synthesis of [28-threonine,31-norleucine]- and [28-threonine,31-leucine]cholecystokinin-pancreozymin-(25-33)-nonapeptide].

The syntheses of two analogues related to the C-terminal nonapeptide amide sequence 25-33 of cholecystokinin-pankreozymin are described. Based on the primary structure of the CCK-PZ-active caerulein and the experiences gained from the methionine replacement with leucine or norleucine in human little gastrin I, the analogues were designed by substituting methionine 28 with threonine, and methionine 31 with leucine and norleucine, respectively. Using a new method for the synthesis of tyrosine-O-sulfate-containing peptides, developed in our laboratory, and applying acid-labile side-chain protection in combination with the benzyloxycarbonyl group, the fully protected nonapeptide amide derivatives Z-Arg(Z2)-Asp(OBut)-Tyr-(SO3Ba1/2)-Thr(But)-Gly-Trp-Leu-Asp(OBut)-Phe-NH2 and Z-Arg(Z2)-Asp(OBut)-Tyr(SO3-Ba1/2)-Thr(But)-Gly-Trp-Nle-Asp(OBut)-Phe-NH2, were obtained. Upon hydrogenolytic and subsequent acidolytic removal of the protecting groups, followed by purification via chromatographic procedures the nonapeptide amides were isolated in satisfactory yields at a high degree of purity. In vivo and in vitro assays showed that a substitution of methionine 31 by norleucine with concomitant replacement of methionine 28 by threonine produced a fully active analogue, whereas for the threonine 28, leucine 31 analogue the pankreozymin-activity was lowered by a factor 10.

Amino Acid Sequence↗

Covalent linking of photoreactive insulin to adipocytes produces a prolonged signal.

The first step of insulin's many cellular functions is specific binding to receptors on the plasma membrane of target cells. The subsequent molecular basis of insulin action, particularly the coupling mechanism(s) involved in transmitting the biological message, remains largely unknown. Our approach to the problem centres on the application of a series of well characterized photo-insulins carrying an aryl-azido or nitro-aryl-azido group in positions A1, B1, B2 or B29 (refs 2--4). Specific binding to membrane components could be demonstrated with B1-, B29- (ref. 5) and A1-photo-insulins as well as a B2-derivative. We now report that lipogenesis is increased to, and maintained at, near-maximal levels for several hours after photoinduced covalent binding of B2- (2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (Napa-DP-insulin) to living adipocytes.

Adipose Tissue↗

[Synthesis of human [11-leucine]minigastrin I--II. Purification of the synthetic tridecapeptide amide and isolation of a side-product (author's transl)].

The preparation of the pure 11-leucine analogue of human minigastrin I, a linear tridecapeptidaemide with full "gastrin activity", from the crude synthetic material obtained after deblocking of the overall protected tridecapeptide amide derivative by means of trifluoroacetic acid, is achieved by ion-exchange chromatography on DEAE-Sephadex A-25 and subsequent partition chromatography on Sephadex G-25. The isolation of a synthetic side-product and its identification as human [10-(Nin-tert-butyl-tryptophan),11-leucine]minigastrin I by means of spectroscopic (UV, fluorescence, 1H-NMR, MS), enzymatic and chromatographic methods is described. The pure tridecapeptide amides human [Leu11]minigastrin I and human [Trp(1'-But)10,Leu11]minigastrin I are isolated in overall yields of 37.4% and 4.6%, respectively (mol-% with regard to the overall protected tridecapeptide amide derivative).

Amides↗

[Side-reactions in peptide synthesis, III. Synthesis and characterization of Nin-tert-butylated tryptophan derivatives (author's transl)].

It was possible to elucidate a side reaction which had long been assumed to occur during the acidolytic cleavage of protecting groups based on a ter-butyl moiety, by synthesizing Nin-tert-butyltryptophan in different ways. It was found to be absolutely identical with a "modified tryptophan" which was isolated after the total synthesis of a gastrin analogue; Nin-tert-butyl-tryptophan peptides are formed as the main products of a tert-butylation reaction, the mechanism of which is not very clear yet. The proof for a Nin-tert-butylation of tryptophan was obtained by spectroscopic methods, in particular mass spectrometry and nuclear magnetic resonance spectroscopy.

Magnetic Resonance Spectroscopy↗

[Side-reactions in peptide synthesis, IV. Charactertzation of C- and C,N-teri-butylated tryptophan derivatives (author's transl)].

Besides Nin-tert-butylated tryptophan derivatives, other tert-butyl-tryptophan analoga are also found as a result of side-reactions accurring during the acidolytic cleavage of protecting groups being based on a tert-butyl moiety. C-mono-substitution could be detected as well as di- and tri-substitution. All these tert-butylated tryptophans could be isolated in a pure form from the tert-butylation mixture either free or as derivatives and could unequivocally be identified in particular by means of mass spectrometry and nuclear magnetic resonance spectroscopy.

Magnetic Resonance Spectroscopy↗

[Direct tert-butylation of tryptophan: preparation of 2,5,7-tri-tert-butyltryptophan (author's transl)].

The tert-butylation of free tryptophan under simulated conditions of acidolytic cleavage of protecting groups being based on a tert-butyl moiety does finally yield the tri-substituted amino acid as a main product, namely 2,5,7-tri-tert-butyltryptophan. The structural elucidation of this substance and other tert-butylated products, which were isolated mostly in a pure form, was achieved by means of spectroscopic methods, in particular mass spectrometry and nuclear magnetic resonance spectroscopy.

Chemical Phenomena↗