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Biomedical subjects

J Engel

Publications and source records attributed to J Engel.

At least 397 records · Page 22Linked to original sources
PubMed1989

The extracellular matrix of Volvox carteri: molecular structure of the cellular compartment.

The extracellular matrix (ECM) of Volvox contains insoluble fibrous layers that surround individual cells at a distance to form contiguous cellular compartments. Using immunological techniques, we identified a sulfated surface glycoprotein (SSG 185) as the monomeric precursor of this substructure within the ECM. The primary structure of the SSG 185 poly-peptide chain has been derived from cDNA and genomic DNA. A central domain of the protein, 80 amino acid residues long, consists almost exclusively of hydroxyproline residues. The chemical structure of the highly sulfated polysaccharide covalently attached to SSG 185 has been determined by permethylation analysis. As revealed by EM, SSG 185 is a rod-shaped molecule with a 21-nm-long polysaccharide strand protruding from its central region. The chemical nature of the cross-links between SSG 185 monomers is discussed.

Amino Acid Sequence↗
PubMed1989

Presence of prolactin-like immunoreactivity and bioactivity in rat spinal cord.

Pursuant to our identification of prolactin-like immunoreactivity (PLI), widely distributed in rat brain, the spinal cord was examined for the presence of this pituitary-hormone-like protein. PLI was present in all spinal cord extracts examined and averaged 500 +/- 53 pg/mg protein. Hypophysectomy, causing a fall in serum prolactin to undetectable levels, was not associated with any change in levels of PLI in spinal cord. Recovery of rat prolactin standards added to spinal cord homogenates was 97.6 +/- 3.9%. When increasing concentrations of spinal cord extract were assayed in a prolactin radioimmunoassay, displacement of rat 125I-Prolactin from antiserum was parallel to that displacement produced by increasing concentrations of rat anterior pituitary standards. Upon subjection to gel permeation chromatography, the elution profiles of immunoreactive prolactin from spinal cord were different from the profiles of anterior pituitary prolactin. In addition to an immunoreactive prolactin peak eluting with pituitary prolactin, spinal cord extracts showed a large void volume peak and late eluting low-molecular-weight materials not seen with anterior pituitary. In the Nb2 lymphoma cell assay, all spinal cord extracts demonstrated prolactin-like bioactivity with a bioactivity/immunoreactivity ratio of 1.05 +/- 0.13. We conclude: (1) PLI, widely distributed in rat brain, is also present in spinal cord; (2) spinal cord prolactin levels are independent of levels in pituitary and peripheral circulation; (3) this immunoreactive prolactin is bioactive, and (4) differing gel permeation chromatographic elution profiles indicate that there may be some molecular differences between pituitary and spinal cord prolactin.

Animals↗
PubMed1989

Ictal localization of temporal lobe seizures with scalp/sphenoidal recordings.

We assessed the reliability and accuracy of scalp/sphenodial recordings for ictal localization by retrospectively analyzing 706 noninvasive ictal recordings from 110 patients who subsequently underwent stereoencephalographic evaluation. Strictly defined unilateral temporal/sphenoidal ictal patterns correctly predicted findings of depth electrode examination in 82 to 94% of cases. These strictly defined predictive patterns could be detected with excellent interrater reliability. The patterns are misleading in only a minority of cases, but cannot be used in isolation for definite ictal localization.

Electroencephalography↗
PubMed1989

Hexadecylphosphocholine, a new ether lipid analogue. Studies on the antineoplastic activity in vitro and in vivo.

Hexadecylphosphocholine (He-PC) is a new compound synthesized according to the minimal structural requirements deducted from studies with other ether lipids. In vitro studies on He-PC revealed remarkable antineoplastic activity on HL60, U937, Raji and K562 leukemia cell lines. In addition, He-PC, applied orally, showed a superior effect in the treatment of dimethylbenzanthracene-induced rat mammary carcinomas when compared to intravenously administered cyclophosphamide. After oral application He-PC was well absorbed from the intestine and metabolized in the liver by phospholipases C and D. During a 5-week treatment no hematotoxic effects were detected. In a clinical pilot study on breast cancer patients with widespread skin involvement, topically applied He-PC showed skin tumor regressions without local or systemic side effects.

Animals↗
PubMed1988

Folding of collagen IV.

Collagen IV dimers of two collagen IV molecules connected by their C-terminal globular NC1 domains were isolated by limited digestion with bacterial collagenase from mouse Engelbreth-Holm-Swarm (EHS) sarcoma tissue. The collagenous domains were only 300 nm long as compared to 400 nm of intact collagen IV but the disulfide bonds in the N-terminal region of the major triple helix were retained. Unfolding of the collagenous domains as monitored by circular dichroism occurred in a temperature range of 30 to 44 degrees C with a midpoint at 37 degrees C. The transition is significantly broader than that of the continuous triple helices in collagens I, II and III, a feature which can be explained by the frequent non-collagenous interruptions in the triple-helical domain of collagen IV. Refolding at 25 degrees C following complete unfolding at 50 degrees C was monitored by circular dichroism, selective proteolytic digestion of non-refolded segments and by a newly developed method in which the recovered triple-helical segments were visualized by electron microscopy. Triple-helix formation was found to proceed in a zipper-like fashion from the C-terminal NC1 domains towards the N-terminus, indicating that this domain is essential for nucleations. For collagen IV dimers with intact NC1 domains the rate of triple-helix growth was of comparable magnitude to that of collagen III, demonstrating that the non-collagenous interruptions do not slow down the refolding process where the rate-limiting step is the cis-trans isomerization of proline peptide bonds. Refolding was near to 100% and the refolding products were similar to the starting material as judged by thermal stability and electron microscopic appearance. Removal of the NC1 domains by pepsin or dissociation of their hexametric structures by acetic acid led to a loss of the refolding ability. Instead products with randomly dispersed short triple-helical segments were formed in a slow reaction. In no case, even when the disulfide bonds in the N-terminal region of the triple-helical domain were intact, was refolding from the N- towards the C-terminus observed. Taken together with results in other collagens, this suggests that C to N directionality might be an intrinsic property of triple-helix folding.

Animals↗
PubMed1988

A substitution of cysteine for glycine 748 of the alpha 1 chain produces a kink at this site in the procollagen I molecule and an altered N-proteinase cleavage site over 225 nm away.

In previous work (Vogel, B. E., Minor, R. R., Freund, M., and Prockop, D. J. (1987) J. Biol. Chem. 262, 14737-14744), we identified a single-base mutation that converted the glycine at position 748 of the alpha 1 chain of type I procollagen to a cysteine in a proband with a lethal variant of osteogenesis imperfecta. In addition to posttranslational overmodification, the abnormal molecules displayed decreased thermal stability and a decreased rate of secretion. An unexplained finding was that procollagen was poorly processed to pCcollagen in postconfluent cultures of skin fibroblasts. Here, we show that the procollagen synthesized by the proband's cells is resistant to cleavage by procollagen N-proteinase, a conformation-sensitive enzyme. Since the only detectable defect in the molecule was the cysteine for glycine substitution, we assembled several space-filling models to try to explain how the structure of the N-proteinase cleavage site can be affected by an amino acid substitution over 700 amino acid residues or 225 nm away. The models incorporated a phase shift of a tripeptide unit in one or both of the alpha 1 chains. The most satisfactory models produced a flexible kink of 30 degrees or 60 degrees at the site of the cysteine substitution. Therefore, we examined the procollagen by electron microscopy. About 25% of the molecules had a kink not seen in control samples, and the kink was at the site of the cysteine substitution.

Binding Sites↗
PubMed1988

Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites.

Intact and proteolytically modified human serpins, alpha 1-proteinase inhibitor, antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor, were compared by circular dichroism, fluorescence spectroscopy, and resistance against unfolding by guanidine HCl. The modified proteins were prepared from the intact and active inhibitors by selective proteolytic cleavage in their reactive site loops and tested for complete loss of activity. Significant differences in the spectral properties between intact and modified inhibitors indicate that a major conformational rearrangement is triggered by the cleavage. This leads to a large increase in conformational stability as demonstrated by large shifts of the transition profiles recorded as a function of guanidine HCl concentration at 20 degrees C by circular dichroism at 220 nm. Intact inhibitors were unfolded in two steps of about equal size centered at 0.8-1.7 and 2.5-3.5 M concentrations of the denaturant, respectively. Under identical conditions modified inhibitors are completely stable, and their denaturation occurs only well above 4 M guanidine HCl in one or two steep transition steps. From the similarity of the spectral changes and shifts in transition profiles for all four serpins studied it is concluded that the conformational changes and stabilization triggered by the modification hit is an important common mechanistic feature of this class of inhibitors. This is supported by the observation that ovalbumin, which is homologous with the serpins but apparently lacks inhibitory activity, exhibits neither spectral changes nor a significant change in stability upon proteolytic modification.

Algorithms↗
PubMed1988

Detection of autocrine motility factor in urine as a marker of bladder cancer.

Cell locomotion is an essential requirement for invasion and metastasis of malignant cells. We have previously described the characterization of a 50-kilodalton autocrine motility factor (AMF), a cytokine that stimulates motility in human tumor cells. In this study, we investigated the elaboration of this factor in vivo by human bladder carcinoma and in vitro by a cultured transitional cell carcinoma (TCC) of the bladder cell line T24P. Urine samples from patients with bladder cancer were assayed for their capacity to stimulate migration of tumor cells. Comparing all TCC cases (22 patients) with all nonmalignant diagnoses (27 patients), we found a statistically significant (P less than .001) difference in the motility values. Invasive TCC cases (15 patients) were significantly (P less than .002) higher in regard to motility values compared with noninvasive TCC cases (8 patients), including one case of carcinoma in situ. In follow-up screening studies evaluating TCC recurrence, the recurrent tumors (9 patients) were higher (P less than .001) in regard to motility values than the tumor-free cases (11 patients). Furthermore, T24P cells showed a dose-dependent motile response to their own serum-free conditioned medium as well as to the AMF present in the urine of TCC patients. This finding is consistent with the source of AMF in the urine of these patients being the cancer itself. An enzyme-linked immunosorbent assay (ELISA) for AMF was also developed. Values determined by ELISA correlated well with the motility values measured separately. These data support the potential usefulness of AMF as a urine marker for bladder TCC.

Adult↗
PubMed1988

Reductive cleavage and reformation of the interchain and intrachain disulfide bonds in the globular hexameric domain NC1 involved in network assembly of basement membrane collagen (type IV).

The formation of collagen IV dimers in the extracellular space requires the association of two C-terminal globular domains giving rise to a large hexameric structure NC1 (Mr = 170,000). NC1 hexamer was purified from collagenase digests of a mouse tumor and several human tissues. It was shown by electrophoresis to consist of two kinds of cross-linked, dimeric segments, Da and Db (Mr about 50,000), and monomeric segments in a molar ratio of about 3:1. In the native hexamers free SH groups were detectable by N-[14C]ethylmaleimide and other sulfhydryl reagents. They account for 4-11% of the total number of cysteine residues with some variations between preparations from different sources and in the distribution between monomers and dimers. Reduction with 10 mM dithioerythritol under non-denaturing condition completely converted dimers into monomers and allowed the alkylation of all twelve cysteine residues present in each monomeric NC1 segment. A monomeric intermediate with four to six free SH groups and a higher electrophoretic mobility than the final product was observed. Generation of this intermediate from dimers Da and Db follows apparently different routes proceeding either directly or through a dimeric intermediate respectively. The time course of conversion is best described by a mechanism consisting of two (Db) or three (Da) consecutive steps with pseudo-first-order rate constants ranging from 0.14 ms-1 to 0.5 ms-1. Glutathione-catalyzed reoxidation of completely reduced NC1 in the presence of 2 M urea results in a product indistinguishable from native material by ultracentrifugation and electrophoresis pattern. The data suggest that in situ formation of NC1 structures is catalyzed by a small fraction (5-10%) of intrinsic SH groups leading to the formation and stabilization of dimers by rearrangement of disulfide bonds.

Animals↗
PubMed1988

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

Animals↗
PubMed1988

Macromolecular organization of natural and recombinant lung surfactant protein SP 28-36. Structural homology with the complement factor C1q.

The macromolecular structure of the pulmonary surfactant apolipoprotein SP 28-36 has been determined. For SP 28-36 isolated from dog lung lavage, a flower bouquet-like hexameric structure with six globular domains connected by short stalks to a common stem was revealed by electron microscopy, using the rotary shadowing technique. This structure is very similar to that published for the subcomponent C1q of the first component of complement C1. The lavage material was compared with the homologous human recombinant SP 28-36 by the same technique. Mostly smaller aggregates like di-, tri- and tetramers as well as very high aggregates were observed. Mild reduction of the recombinant material revealed the lollipop-shaped monomers composed of a globular domain and a tail with a discrete kink in the middle portion. The collagenous nature of the tail was demonstrated by circular dichroism spectroscopy. This implies that the mammalian expression system assembles the monomeric subunits correctly. Assembly into the hexameric structures, however, does not proceed quantitatively.

Animals↗
PubMed1988

A mass spectral study of cyclophosphamide concerning a thermally induced rearrangement reaction.

The electron impact mass spectra of cyclophosphamide (1) are very sensitive towards experimental conditions in view of the kind of sample handling, the type of mass spectrometer used and the temperature of evaporation. The reason for this phenomenon is the elimination of HCl from the molecular ion by a specific 1,5-hydrogen transfer yielding an ion at m/z 224 which is structurally related to the bicyclic compound 4 with its typical fragment ions at m/z 175 and 147. Thermal excitation of the sample increases the intensity of this fragmentation pathway. The fragmentation pattern of 1 and the thermally induced rearrangement reaction has been elucidated by means of isotopic labelling, high-resolution data, metastable ion analysis and some tandem mass spectrometric experiments. Various samples of 1 monohydrate and its commercially available preparations, which are triturates with sodium chloride, differing in the crystal size distribution, showed nearly identical mass spectra on two different magnetic mass spectrometers, provided that the materials were introduced as solids under careful control of the evaporation temperature. The fragmentation via m/z 224 prevails in case of non-crystalline, pre-dissolved samples on one of the instruments used which might be explained by a differing construction of the ion source and the sample cup holder. The conclusions of Mruzek et al. concerning different proportions of stereoisomers in pharmaceutical preparations of 1 lack any analytical evidence.

Chemical Phenomena↗
PubMed1988

Effect of clonidine on ultrasonic vocalization in preweaning rats.

The present study was undertaken to investigate the involvement of the noradrenergic neurotransmission system in the ultra sonic callings emitted by rat pups separated from their mother and exposed to cold stimulation. The investigation was primarily performed by help of agents selectively affecting the alpha-adrenoceptors: the alpha 2-agonist clonidine, the alpha 1-antagonist prazosin and the alpha 2-antagonist idazoxan. Clonidine dose-dependently stimulated the amount of ultra sonic vocalization, an effect not solely dependent upon the effect of clonidine on body temperature. In a developmental study it was found that clonidine uniformly stimulated crying at all ages from 4 days of age up to 18 days of age, that is during the whole preweaning period. Clonidine stimulated ultrasonic crying in rat pups, devoid of presynaptic catecholamine (CA) neurons by combined pretreatment with the monoamine depletor, reserpine, and the inhibitor of CA-synthesis, alpha-methyl-tyrosine. This finding suggested that the stimulating effect of clonidine on ultrasonic vocalization was mediated by postsynaptic adrenoceptors. In pups, 12 days of age, idazoxan blocked the effect of cold stimulation on ultra sonic crying, suggesting that alpha 2-adrenoceptors, presumably postsynaptic ones, mediated this kind of stimulation. Idazoxan also antagonized the effect of clonidine, but only at a dose effective also in control pups. Prazosin had no effect on cold-stimulated crying, but antagonized the effect of clonidine, suggesting that the effect of clonidine was also mediated by alpha 1-receptors. At 18 days of age, prazosin no longer antagonized the effect of clonidine, whereas the antagonizing action of idazoxan was reinforced. The age-dependent variation in responsiveness to the adrenergic drugs suggest maturational changes in the function of the CA-system occurring between 12-16 days of age.

Adrenergic alpha-Antagonists↗
PubMed1988

Possible involvement of noradrenergic descending pain-modulating pathways in the mode of antinociceptive action of flupirtine, a novel non-opioid analgesic.

These experimental studies were conducted to obtain information about the antinociceptive action of flupirtine within the central nervous system. Flupirtine dose-dependently increased pain threshold in the electrostimulated pain test in mice. Its antinociceptive activity was attenuated by simultaneous administration of the noradrenergic alpha 1/alpha 2-antagonist yohimbine and alpha 2-antagonist idazoxane. By contrast, the analgesia induced by codeine or morphine was not influenced by alpha 2-adrenergic antagonists at all. A striking resemblance could be observed in the pharmaco-EEG of freely moving rats treated with clonidine and flupirtine, respectively. The present results are consistent with the hypothesis that the noradrenergic descending pain-modulating system might be involved in the antinociceptive mode of action of flupirtine.

Aminopyridines↗