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A Betz

Publications and source records attributed to A Betz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transient, phorbol ester-induced DOC2-Munc13 interactions in vivo.

Munc13-1 and DOC2 have been implicated in the regulation of exocytosis. Here we demonstrate in vivo that these two proteins undergo a transient phorbol ester-mediated and protein kinase C-independent interaction, resulting in the translocation of DOC2 from a vesicular localization to the plasma membrane. The translocation of DOC2 is dependent upon the DOC2 Munc interacting domain that binds specifically to Munc13-1, whereas the association of DOC2 with intracellular membranes is dependent on its C2 domains. This is the first direct in vivo demonstration of a protein-protein interaction between two presynaptic proteins and may represent a molecular basis for phorbol ester-dependent enhancement of exocytosis.

Alkaloids↗
PubMed1999

Cloning, sequence analysis, and expression of the Pseudomonas putida 33/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase.

1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).

Amino Acid Sequence↗
PubMed1999

Differential expression of two novel Munc13 proteins in rat brain.

Munc13-1, a mammalian homologue of Caenorhabditis elegans unc-13p, is a presynaptic phorbol ester receptor that enhances neurotransmitter release. In the present study we analysed the regional, cellular and subcellular expression patterns in rat of two novel Munc13 proteins, Munc13-2 and Munc13-3. We demonstrate by hybridization in situ that Munc13-1 mRNA is expressed throughout the brain, whereas Munc13-2 mRNA is preferentially present in rostral brain regions, and Munc13-3 mRNA in caudal areas. In an analysis of subcellular brain fractions with isoform-specific antibodies, we show that the novel Munc13 proteins are enriched in synapses. Immunocytochemical examination of rat cerebellar sections indicates that Munc13-3, like Munc13-1, is concentrated in presynaptic terminals. Our results characterize Munc13 proteins as a family of neuron-specific, synaptic molecules that bind to syntaxin, an essential mediator of neurotransmitter release. Munc13-2 and Munc13-3 are expressed in a complementary fashion and might act in concert with Munc13-1 to modulate neurotransmitter release.

Amino Acid Sequence↗
PubMed1999

An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.

We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.

Adrenal Glands↗
PubMed1998

Regions remote from the site of cleavage determine macromolecular substrate recognition by the prothrombinase complex.

The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, alpha-thrombin, was a linear competitive inhibitor of prethrombin 2 activation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and alpha-thrombin compete for binding to the exosite without restricting access to the active site of factor Xa within prothrombinase. Inhibition by alpha-thrombin was not altered by saturating concentrations of low molecular weight heparin. Furthermore, proteolytic removal of the fibrinogen recognition site in alpha-thrombin only had a modest effect on its inhibitory properties. Both alpha-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp148 and separated into component domains. The C-terminal-derived zeta2 fragment retained the ability to selectively inhibit macromolecular substrate cleavage by prothrombinase, while the zeta1 fragment was without effect. As the zeta2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is achieved through substrate regions, distinct from the fibrinogen recognition or heparin-binding sites, and spatially removed from structures surrounding the scissile bond.

Animals↗
PubMed1998

Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release.

Munc13-1, a mammalian homolog of C. elegans unc-13p, is thought to be involved in the regulation of synaptic transmission. We now demonstrate that Munc13-1 is a presynaptic high-affinity phorbol ester and diacylglycerol receptor with ligand affinities similar to those of protein kinase C. Munc13-1 associates with the plasma membrane in response to phorbol ester binding and acts as a phorbol ester-dependent enhancer of transmitter release when overexpressed presynaptically in the Xenopus neuromuscular junction. These observations establish Munc13-1 as a novel presynaptic target of the diacylglycerol second messenger pathway that acts in parallel with protein kinase C to regulate neurotransmitter secretion.

Amino Acid Sequence↗
PubMed1998

Women's safety in recovery: group therapy for patients with a history of childhood sexual abuse.

The literature on group therapies for women with histories of childhood sexual abuse has focused on outpatient treatments. A model of group treatment for inpatients and partial hospital patients is described here. "Women's Safety in Recovery" is a first-stage trauma recovery group that promotes mastery of current life stressors and prevents regression through the use of psychoeducation about abuse effects, the practice of problem-solving skills, and supportive, topic-focused discussion. The group is structured in three one-week modules that patients can begin or leave at any time.

Adaptation, Psychological↗
PubMed1997

Exosites determine macromolecular substrate recognition by prothrombinase.

The prothrombinase complex, composed of factor Xa and factor Va assembled on a membrane surface, catalyzes the proteolytic formation of thrombin during blood coagulation. The molecular basis for the macromolecular substrate specificity of prothrombinase is poorly understood. By kinetic studies of prethrombin 2 cleavage by prothrombinase in the presence or absence of fragment 1.2, we show that occupation of the active site of the catalyst by inhibitors or alternate peptidyl substrates does not alter the affinity for prethrombin 2. Productive recognition of the macromolecular substrate therefore results from an initial interaction at enzymic sites (exosites) distinct from the active site, which largely determines substrate affinity. This interaction at exosites is evident even in the absence of activation peptide domains responsible for mediating the binding of the substrate to membranes or factor Va. Interactions at the active site with structures surrounding the scissile bond then precede bond cleavage and product release. The second binding step, which appears unfavorable, does not affect substrate affinity but contributes to the maximum catalytic rate. Therefore, binding specificity of prothrombinase for the macromolecular substrate is determined by exosites on the enzyme. We show that competitive inhibition of prethrombin 2 cleavage can be accomplished by interfering with the exosite binding step without obscuring the active site of the enzyme. These findings suggest limitations to the common approach of inferring the basis of factor Xa specificity with active site mutants or the targeting the active site of factor Xa with reversible inhibitors for therapeutic purposes. The achievement of distinctive macromolecular substrate specificities through exosite interactions and modulation of maximum catalytic rate through binding steps may also underlie the reactions catalyzed by the other coagulation complexes containing trypsin-like enzymes.

Animals↗
PubMed1997

Direct interaction of the rat unc-13 homologue Munc13-1 with the N terminus of syntaxin.

unc-13 mutants in Caenorhabditis elegans are characterized by a severe deficit in neurotransmitter release. Their phenotype is similar to that of the C. elegans unc-18 mutation, which is thought to affect synaptic vesicle docking to the active zone. This suggests a crucial role for the unc-13 gene product in the mediation or regulation of synaptic vesicle exocytosis. Munc13-1 is one of three closely related rat homologues of unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. In the present study, we demonstrate by three independent methods that the C terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homologue Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin.

Animals↗
PubMed1997

Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.

The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.

Animals↗
PubMed1997

[Possibilities of osteotomy. Osteotomy and corticotomy techniques].

Corrective measures on the bone undertaken after the fusion of epiphyses are only possible through an initial break in its continuity. This paper deals with the different methods of osteotomy available to the orthopaedic or trauma surgeon. After introducing the terminology of the so-called "osteotomy", the vascularity of the bone, special features of the various osteotomy site, the different indications, and particular details of the operative procedures are discussed. Special emphasis is placed on minimally invasive techniques and osteotomies in the framework of callus distraction. In addition to established procedures a new sawing technique for the Küntscher's closed osteotomy is described.

Bone Lengthening↗
PubMed1997

A fully implantable motorized intramedullary nail for limb lengthening and bone transport.

This article describes an intramedullary nail that contains a fully implantable motorized programmable sliding mechanism for limb lengthening and bone transport that reduces the risk of infection, discomfort, and scarring usually associated with the external fixators used for the same purpose. Twelve patients were treated surgically with the new system. Eleven patients had unilateral femur shortening between 3 and 7.5 cm, and one patient had a 12-cm defect after tumor resection. In all patients with femur shortening the leg length discrepancy was corrected completely. In the case of bone defect the segment transport worked well without any problems. There was no infection and no axial deformity. Immediately after chemotherapy, delayed bone formation was seen. In two early cases of limb lengthening a technical problem led to replacement of the motor.

Adolescent↗
PubMed1996

Characterization of vitellogenin from rainbow trout (Oncorhynchus mykiss).

The nucleotide sequence of the vitellogenin cDNA from the rainbow trout Oncorhynchus mykiss was determined. Analysis of the deduced amino acid sequence (1659 residues) places the lipovitellin I, phosvitin and lipovitellin II domains between amino acids 16 to 1088, 1089 to 1145 and 1146 to 1659, respectively. The general structure is similar to other vertebrate vitellogenins except for the serine rich phosvitin domain which is the shortest identified so far in vertebrates (57 amino acids), being 2 to 4 times smaller than in other species. Sequence comparisons between vertebrate and invertebrate vitellogenins as well as with distantly related proteins allowed to identify two short amino acid motifs particularly well conserved, RGILN and TCGLCG in lipovitellin I and II domains, respectively, and strongly suggest that the lipovitellin II domain is involved in protein interactions via disulfide bridge formation.

Amino Acid Sequence↗
PubMed1996

[Intramedullary nailing in callus distraction].

Intramedullary nailing is an established fixation method in fracture treatment as well as in deformity-correction procedures on tubular bones. The interlocking nail has increased the potential for employing the intramedullary nail in the treatment of fractures in metaphysial regions. Recent developments are biased towards the use of small-diameter unreamed nails, preferably made of titanium. Callus distraction, the controlled continuous shifting of corticotomized bone, leads to new bone formation and has been known since the beginning of this century. This method, however, gained wide acceptance and popularity through the work of Ilizarov. This paper describes a fusion of the two methods into a very promising management concept for substantial bone defects resulting from trauma or resection of bone tumors, as well as for the correction of congenital or acquired uni- or bilateral differences in length of the extremities. Various systems such as the combination of callus distraction by external fixation over an intramedullary nail and a fully implantable system are presented.

External Fixators↗
PubMed1996

[Perspectives in callus distraction].

Callus distraction is an enrichment of the surgical orthopaedic way of reconstructing bone defects, but it is also an effective method of lengthening limbs. The biological facts correspond to our knowledge about fracture healing. Concerning apparatures, further development is needed. In certain cases the ring fixator is the method of choice, but in most indications it is not necessary. Every effort should be made to adapt modern technologies, including fully implantable systems, to the "biological method" of callus distraction, whenever possible.

Bone Lengthening↗
PubMed1995

[Contralateral replantation after bilateral traumatic lower leg amputation. Case report with 6 year follow-up].

A 66-year-old patient attempted suicide by jumping in front of a train. The lower extremities were amputated at different levels. On the right side, there was a complete amputation within the distal third of the lower leg. Proximal to the amputation site, there was an extensive soft-tissue and bone defect. On the left side, there was a crush injury of the tarsal and mid-tarsal bones. The left lower leg showed only few injuries. An ipsilateral (anatomical) replantation was not possible. In order to save one lower extremity, we decided to carry out a cross-over (contralateral) replantation of the right foot to the left lower leg. After a follow-up of six years, the patient is able to walk well with her prosthesis on the right side and the right foot hooked up to the left lower leg. Functionally, this treatment (cross-over replantation-one-side prosthesis of the lower leg) is much better than the prosthesis on both extremities, as the result has shown. Also from a psychological point of view, it seems to be better for the patient to preserve one extremity even with a cross-over replanted foot.

Aged↗
PubMed1995

[Fracture healing after intramedullary nailing of simple tibial shaft fractures. A clinical comparison of reamed and unreamed procedures].

From January 1990 to June 1993, 56 patients with simple tibial shaft fractures were treated in the Surgical Department of the University/Municipal Hospital in Munich by primary intramedullary nailing, and 44 of these patients were followed up. The results in 17 who underwent unreamed intramedullary nailing (UTN) were compared with those in 27 in whom reamed procedures (RTN) were applied. There was no difference between the two groups in age, fracture type and localization. Soft tissue trauma prevailed, with 35% I degrees open fractures in the UTN group (RTN group, 3%). UTN patients were operated on an average of 45 h after trauma, and RTN patients, 5 days after trauma. Both groups showed about the same proportion of good and very good results (criteria of Johner and Wruhs), with 83% in the UTN group and 84% in the RTN group. The rate of complications was the same in both groups (11%), and we did not find any kind of infection. Two complications requiring revisions (nonunion, perforation of the nail) after UTN stress the importance of two-dimensional barring in the main fragments (especially when close to the metaphysis) and of reduced weight-bearing for 6 weeks after the operation. The slightly greater intramedullar instability after UTN did not cause a higher rate of nonunions or of fracture healing in a wrong position than RTN. The X-ray findings showed beginning osseous reunion after 13 weeks in the UTN group. This corresponds to earlier painless full weight-bearing after an average of 9.7 weeks, as against 12 weeks in the RTN group.

Adolescent↗