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J Engel

Publications and source records attributed to J Engel.

At least 19 recordsLinked to original sources
PubMed1992

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Polymerization of pNcollagen I and copolymerization of pNcollagen I with collagen I. A kinetic, thermodynamic, and morphologic study.

Previous observations established that pNcollagen III copolymerized with collagen I and decreased the diameter of the fibrils formed (Romanic, A.M., Adachi, E., Kadler, K.E., Hojima, Y., and Prockop, D.J. (1991) J. Biol. Chem. 266, 12703-12709). Here, procollagen I alone or mixtures of procollagen I and pCcollagen I were incubated with procollagen C-proteinase to generate pNcollagen I or mixtures of pNcollagen I and collagen I. The results confirmed previous reports that pNcollagen I assembles into sheet-like structures. They also demonstrated that polymerization of pNcollagen I exhibits a lag period and propagation phase similar to those seen with other protein self-assembly systems. In addition, the results demonstrated that pNcollagen I formed true copolymers with collagen I in that the presence of pNcollagen I increased the lag time, decreased the propagation rate, and increased the concentration of collagen I in solution at equilibrium. Copolymerization of pNcollagen I with collagen I, however, differed in two features from copolymerization of pNcollagen III with collagen I. One was that, in confirmation of previous work, copolymerization of pNcollagen I with collagen I markedly altered the circularity of the fibrils formed. The second difference was that the copolymerization increased the concentration in solution at equilibrium of pNcollagen I whereas copolymerization with collagen I was previously shown to decrease the concentration in solution of pNcollagen III. The increase in concentration in solution of pNcollagen I was explicable either by the assembly of soluble oligomers of pNcollagen I and collagen I, or by subtle changes in the activities of pNcollagen I and collagen I in the solid-phase. Comparison with previous data with pNcollagen III indicated that although pNcollagen I and pNcollagen III copolymerize with collagen I, there are marked differences in the two kinds of copolymers.

Collagen↗
PubMed1992

Genes involved in tumor invasion and metastasis are differentially modulated by estradiol and progestin in human breast-cancer cells.

Invasion of basement membranes by cancer cells is a critical step in metastasis, which requires the coordinated expression of specific genes such as laminin receptors and metalloproteinases. Estradiol and progesterone modulate the clinical progression of steroid-sensitive breast cancers; however, little is known about the molecular regulation of the invasive phenotype by these hormones. We therefore examined the effects of 10 nM estradiol and/or 10 nM progestin R5020 on the expression of 2 non-integrin laminin binding proteins, the 67-kDa laminin receptor (67LR) and HLBP31 as well as the 72-kDa type-IV collagenase (MMP-2) and its inhibitor, TIMP-2, in steroid-receptor-positive (T47D and MCF-7) and -negative (MDA-MB 231) human breast-cancer cells. The relative steady-state level of 67LR mRNA was increased 2- to 3-fold by estradiol in both MCF-7 (p < 0.001) and T47D (p < 0.001) cells, also by R5020, alone or in combination with estradiol, in T47D cells (p < 0.001) and to a much less extent in MCF-7 cells. HLBP31 mRNA and protein levels were increased 2- to 3-fold (p < 0.001) by R5020 alone or in combination with estradiol, but not by estradiol alone. None of the steroid treatments affected the expression or activity of MMP-2. Interestingly, however, TIMP-2 mRNA levels and protein expression in MCF-7 and T47D cells were 50% down-regulated (p < 0.001) by treatment with R5020 or R5020 plus estradiol, but not by treatment with estradiol alone. None of these genes were modulated in steroid-independent MDA-MB231 cells. The data suggest that estradiol and progesterone might act as coordinators regulating specific genes in the steroid-sensitive breast-cancer cell, leading to the acquisition of the metastatic phenotype.

Breast Neoplasms↗
PubMed1992

Recombinant laminin B1 chains exhibit intact short-arm domains but do not form oligomeric molecules.

The human laminin B1 chain has been produced in the baculovirus expression system in sufficient amounts for biochemical and functional studies. A full-length cDNA, which was constructed of four partially overlapping clones and verified by in vitro transcription and translation to be functional, was cloned into the transfer vector pVL1392 behind the polyhedrin promoter. The recombinant construct was incorporated by in vivo homologous recombination into the genome of the wild-type baculovirus, Autographa californica nuclear polyhedrosis virus. Infection of Spodoptera frugiperda cells (Sf9) with the recombinant virus resulted in the expression of the recombinant B1 chain (recB1) in these insect cells. The recB1 was found to be synthesized in two forms with apparent molecular masses of 220 kDa and 200 kDa. The 220-kDa form is an N-glycosylated form of recB1, because it was not present in cultures containing tunicamycin, an inhibitor of N-linked glycosylation. The recB1 accumulated inside the cell and only a small portion of it was secreted into the culture medium. Thus purification had to be started from the cell extract in order to obtain reasonable amounts of the protein. About 500 micrograms was obtained from a 500-ml culture with three steps of chromatography, concavalin A, DEAE-Sepharose and Mono Q anion-exchange chromatography. Only the glycosylated form was purified. The recB1 was found to be sensitive to degradation during the purification, because two proteolytic forms of about 180 kDa were present in every preparation. The accumulation of recB1 inside the cell was possibly due to the lack of correct assembly. Electron microscopy studies showed that the short arm part had a native or near-native structure, but the C-terminal heptad repeat domain had not folded correctly and did not exist in an alpha-helical structure, as it does in native laminin. Electron microscopy and cross-linking studies further revealed that recB1 was a monomeric protein. It was also shown to be unable to oligomerize in vitro, suggesting that the B1 chain is not designed to form homo-oligomers. Finally, cell attachment assays were carried out, but the native recB1 appeared to be inactive in these assays.

Animals↗
PubMed1992

Proteoglycans from the swarm rat chondrosarcoma. Structure of the aggregates extracted with associative and dissociative solvents as revealed by electron microscopy.

Proteoglycan aggregates were extracted from Swarm rat chondrosarcoma tissue in the native state and compared with proteoglycan aggregates isolated dissociatively with 4 M guanidine HCl. Purified aggregates were examined with a variety of electron microscopic techniques. In some cases they showed a structure of the central filament identical to that of the link-stabilized central filament observed in earlier experiments where the separated constituents were allowed to reconstitute (Mörgelin, M., Paulsson, M., Hardingham, T. E., Heinegård, D., and Engel, J. (1988) Biochem. J. 253, 175-185). The tight packing of proteoglycan monomers along the hyaluronate with a minimum distance of 12 nm between adjacent E1 strands also could thus be confirmed for never dissociated aggregates. The results therefore show that the organization of proteoglycan aggregates assembled in vitro from the participating molecules is representative for conditions in situ. An additional structural type of central filament was observed in the preparations. This contained long stretches of free hyaluronate interspaced by short stretches of central filament with condensed arrays of link protein-proteoglycan. Chemical cross-linking in combination with low shear electron microscopical techniques showed that this discontinuous central filament structure is not an artifact of specimen preparation. The addition of suprastoichiometric amounts of exogenous link protein did not affect the central filament structure with the low packing density. Densely and loosely packed types of central filament were isolated in varying relative amounts with different associative and dissociative solvents.

Animals↗
PubMed1992

Alpha-2-adrenoceptor sensitivity in early alcohol withdrawal.

Growth hormone (GH), blood pressure, and pulse rate responses to clonidine (100 micrograms IV) were studied three times during the first week of alcohol withdrawal in 19 alcohol-dependent patients. Fifteen healthy men were used as controls. The results suggest reduced sensitivity of the alpha-2-adrenoceptors involved in GH secretion for at least 1 week after the end of alcohol intake. In contrast, very short-lasting subsensitivity was found in the alpha-2-adrenoceptors regulating blood pressure.

Adult↗
PubMed1992

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.

Amino Acid Sequence↗
PubMed1992

Laminin chain assembly by triple and double stranded coiled-coil structures.

We have previously provided evidence that laminin assembly occurs by the specific interaction of the alpha-helical domains of the A, B1, and B2 chains, located within the long arm of the molecule (Hunter, I., Schulthess, T., Bruch, M., Beck, K., and Engel, J. (1990) Eur. J. Biochem. 188, 205-211). Recent evidence for noncoordinate synthesis of the laminin chains, and in particular, the absence of the 400-kDa A chain from laminins produced by a number of cell types, has led us to examine the molecular mechanism of laminin assembly using the isolated A and B1-B2 chains of laminin fragment E8. E8A shows little tendency to self-associate, and when renatured from urea forms globular structures with little detectable alpha-helix. In contrast, E8B1-B2 renatures to form rod-like molecules, 30 nm in length. The rod-like structure, high alpha-helix content, and sharp thermal transition indicate that they are double stranded coiled coils. When mixed in equimolar amounts, E8A and E8B1-B2 renature to form molecules which are biochemically and ultrastructurally indistinguishable from native E8. If E8A and E8B1-B2 are renatured separately and mixed at a 1:1 molar ratio, they also form E8 molecules. These results suggest a mechanism of laminin assembly which involves the formation of a double coiled-coil B1-B2 intermediate with which the A chain subsequently interacts to form a triple coiled-coil laminin molecule. In addition, our results indicate that isoforms consisting of the B1 and B2 chains only would form stable "laminin-like" structures.

Circular Dichroism↗
PubMed1992

Electron microscopy of native cartilage oligomeric matrix protein purified from the Swarm rat chondrosarcoma reveals a five-armed structure.

Cartilage oligomeric matrix protein was isolated in the native state from the Swarm rat chondrosarcoma. A crucial step was its selective extraction with EDTA-containing buffer. The purified protein was subjected to electron microscopy using rotary shadowing and negative stain. The images allowed the construction of a structural model. The bouquet-like protein consists of five 28-nm-long arms containing a peripheral globular domain, a flexible strand, and a central assembly domain, where the five arms meet in a cylindrical structure.

Animals↗
PubMed1992

[Lateral arm free flap transfer to cover traumatic hand injuries].

The advantages of free flaps over distant pedicled flaps in hand reconstruction are well documented. They often require only a single operation, do not depend on local conditions of the wound and permit early physiotherapy. The lateral arm free flap is essentially suitable for reconstruction of the hand. It is thin, potentially sensate, and gives nearly perfect skin color and texture match. An additional technical advantage is the possibility of elevating the lateral arm free flap with tourniquet control at the same surgical field as the recipient site. We describe 3 cases of traumatic damage to skin and soft tissue of the hand, in which reconstruction was by both elective and emergency lateral arm, free flap transfer. They demonstrate the merits of this method of reconstruction.

Adult↗
PubMed1992

[Enantioselective biotransformation of a new theophylline derivative].

Reductive amination of 4 with (RS)-2 is an improved method to obtain 7-((RS)2-((RS)-1-Methyl-2-phenyl-ethylamino)propyl)-theophylline (3) (ratio of diastereomers 1:1) compared with SN-displacement of proxyphyllintosylate with (RS)-2 (ratio of diastereomers 9:1). Due to steric hindrance 3 is no noteworthy prodrug for the intermediate formation of (RS)-2 in rats. The enantioselectivity of 2 after a single oral dose of 3 is determined in 24-h urine by HPLC on a Cyclobond-column using the isoindole derivatives, formed from o-phthalaldehyde, thioglycolic acid, and 2. The R(-)-amphetamine isomer is formed to a lesser amount (34.8%, range 32-37%) than the S(+)-isomer (65.2%, range 63-68%).

Animals↗
PubMed1992

Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

A large portion of the N-terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1-1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9-N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease-sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9-N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9-N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9-N2 for the triple helical region of collagen VI suggesting a role of the N-terminal globule in the self-assembly of microfibrils. No or only little binding was, however, observed to fibril-forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9-N2 was also an inactive substrate for cell adhesion.

Amino Acid Sequence↗
PubMed1992

Treatment of experimental DMBA induced mammary carcinoma with Cetrorelix (SB-75): a potent antagonist of luteinizing hormone-releasing hormone.

Cetrorelix, (Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10)-LHRH (SB-75) is a new highly potent antagonist of LH-RH. In the model of DMBA-induced mammary carcinoma, this antagonist was very effective in reducing tumor mass. A rapid decrease in tumor weights to levels below 0.1 g total tumor mass was achieved with 300 micrograms/kg given sc. daily for 14 days. The weights of uteri and ovaries were reduced to about 40-50% of control values. In all treated rats the estrus cycle was interrupted and the animals remained in a state of anestrus. Microscopically, the effects of Cetrorelix on the tumors were characterized by a loss of mitotic activity, marked regression with apoptosis, an increase of stroma and differentiation towards a normal mammary architecture. On the basis of a dose-response curve, a dose of 100 micrograms/kg/d of Cetrorelix was determined as sufficient for a full antitumor response. Large DMBA-tumors with total tumor mass of about 6 g could also be treated very effectively with a dose of 100 micrograms/kg/d. To achieve a complete tumor regression, the treatment had to last 34 days. After the cessation of treatment with 100 micrograms/kg/d and regrowth of the tumors the animals were treated with the agonist Decapeptyl (Trp6-LHRH) using a dose of 50 micrograms/rat/d for 14 days. Again, the tumors responded well and regressed within 10 days. The treatment with an overlapping dose schedule of Cetrorelix and Decapeptyl showed a continuous antitumor response. A transient stimulation of tumor growth by the LH-RH agonist was not observed under these experimental conditions. In ovariectomized rats bearing DMBA-tumors, treatment with Cetrorelix and estradiol, produced no tumor growth inhibition as compared to estradiol control group, indicating that there is no estrogen nullifying effect of this antagonist on tumor cells in this model. On the basis of these results, Cetrorelix is a highly effective antitumor agent in this breast cancer model, which might also be useful under clinical conditions.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

A health-related quality of life instrument for patients evaluated for epilepsy surgery.

The goals of surgery in treating intractable epilepsy are to eliminate seizures and improve quality of life. This report describes the development of the Epilepsy Surgery Inventory (ESI)-55, a 55-item measure of health-related quality of life for epilepsy patients. The ESI-55 includes the following scales (number of items in parentheses): health perceptions (9), energy/fatigue (4), overall quality of life (2), social function (2), emotional well-being (5), cognitive function (5), physical function (10), pain (2), and three separate scales of role limitations due to emotional, physical, or memory problems (5 items each). Also included is one change in health item. The ESI-55 was completed by 89% of 224 adults who had undergone a protocol evaluation for epilepsy surgery since 1974. Alpha internal consistency reliability coefficients ranged from 0.76 to 0.88 except for social function (alpha = 0.68). Multitrait scaling analyses supported item discrimination across scales. Factor analysis confirmed previously identified mental and physical health factors, and yielded a third factor defined by cognitive function and role limitations scales. Construct validity was supported by correlations of the ESI-55 with a mood profile instrument. Analysis of ESI-55 scale scores by seizure classification showed that the 44 patients who were seizure-free following surgery scored higher than did 55 patients who continued to have seizures (P less than 0.05 for all comparisons); 43 patients having seizures without loss of consciousness scored in between. Results of this study indicate that the ESI-55 is reliable, valid, and sensitive to differences in seizure status.

Adult↗