Jürgen Hoppe

The proton conducting F0-part of bacterial ATP synthases

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Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine‐regulate neurotransmitter release by phosphorylation‐dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock‐out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock‐out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified ‘model’ synapse of the larval nerve muscle preparation revealed no difference between wild‐type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild‐type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Mitogenic effect of platelet-derived growth factor in human glomerular mesangial cells: modulation and/or suppression by inflammatory cytokines

Glomerular mesangial cell proliferation constitutes a frequent pathological alteration in glomerulonephritis. In addition to platelet‐derived growth factor (PDGK) inflammatory cytokines such as IL‐1. IL‐6 or tumour necrosis factor‐alpha (TNF‐α) have been proposed to have mitogenic activity for mesangial cells. A model was therefore established in which human mesangial cells (HMC) could be reversibly growth‐arrested for prolonged times in serum‐free medium without suffering irreversible functional or morphological changes. In this model 24 h stimulation with rhPDGF‐BB induced an increase of the 3H‐thymidine incorporation of 1190.280 (50 ng/ml) %± s.e.m. of medium control. Less growth induction was noted after stimulation with 50 ng ml rhPDGF‐AB (925± I26) or rhPDGF‐AA (575 ± 24%). Northern analysis confirmed the presence of both α and β‐PDGF receptor subunit mRNA in growth‐arrested HMCs. rhlL‐lα, rhlL‐1β, rhTNF‐α or rhIL‐6 at various doses and times, despite increasing cellular PGE2‐release, did not induce significant proliferation in HMCs. Inhibition of PGE2‐release did not change the lack ol mitogenicity of lL‐l, TNF‐α or lL‐6. IL‐6 did not alter the mitogenic response of the cells towards PDGF. In contrast, both IL‐lα and lL‐lβ (5 ng/ml) induced a delay but not augmentation of the PDGF growth response. This delay could be reversed by the concomitant addition or recombinant IL‐6 or of anti‐lL‐1 antibody but not by inhibition of prostaglandin synthesis. High doses of TNF‐α suppressed PDGF‐induced proliferation. These data suggest that in growth‐arrested HMCs inflammatory cytokines have a growth‐modulating or ‐suppressive rather than (co‐)mitogenic effect while PDGF‐BB and‐AB and to a lesser degree PDGF‐AA are potent mitogens. The findings support the notion that the control of HMC proliferation in pathological situations depends on a complex network of interacting stimuli.

The platelet-derived growth factor isomers, PDGF-AA, PDGF-AB and PDGF-BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms

The effect of human recombinant platelet‐derived growth factor (PDGF) isoforms, (r)PDGF‐AA, PDGF‐AB and PDGF‐BB, on contractility of rat aortic rings as well as on intercellular free Ca2+ ([Ca2+]i), intracellular pHi (pHi) and thromboxane A2 (TXA2) formation in cultured vascular smooth muscle cells (VSMC) was examined. PDGF‐BB behaved similar to PDGF‐AB and both have features characteristic of conventional vasocontrictor‐agonists that directly increase [Ca2+]i, activate the Na+/H+ exchanger, stimulate the TXA2 formation, and induced contraction in VSMC whereas PDGF‐AA induced contraction without increasing of [Ca2+]i, pHi, and TXA2 formation.

Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio‐Gel P‐30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.

An Asp—Asn substitution in the proteolipid subnit of the ATP-synthase from Escherichia coli leads to a non-functional proton channel

A carbodiimide binding proteolipid subunit has been identified in all ATP synthase complexes [l-8]. Due to its oligomeric form it constitutes the major part of the membrane factor Fu which in Escherichia coli is composed of three different subunits a, b, c (proteolipid) [7,8]. Dicyclohexylcarbodiimide binds covalently to the proteolipid leading to an inhibition of the proton translocation through the membrane [l-8]. Sequence analysis of ATP synthase proteolipid subunits from various organisms (Neurospora crassa, Saccharomyces cerevisiae, bovine heart, chloroplasts (spinach), mastigocladus laminosus (blue green algae) and bacteria (E. coli, PS-3, Bacillus acido caldarius) revealed an invariant acidic amino acid located in a very hydrophobic stretch of amino acids which is selectively modified by the hydrophobic reagent dicyclohexylcarbodiimide [9-151. DCCD reactive aspartyl residue is exchanged by an asparagine. This in sterical terms smallest alteration leads to a non-functional proton channel. Thus, an acidic amino acid side chain at this position seems to be essential.

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.

The induction of early response genes in rat smooth muscle cells by PDGF-AA is not sufficient to stimulate DNA-synthesis

The effect of the three platelet‐derived growth factor (PDGF) isoforms AA, AB and BB on the induction of the early growth response genes c‐fos, egr‐1 and c‐myc mRNA in vascular smooth muscle cells from rat was compared with their respective mitogenic potency. The three PDGF isoforms strongly stimulated the induction to a similar extent. In contrast, PDGF‐AB and ‐BB provoked a marked DNA synthesis whereas PDGF‐AA exerted only a poor mitogenic effect in smooth muscle cells. PDGF‐AA‐stimulated receptor autophosphorylation was not detectable in comparison with the strong effect elicited by PDGF‐AB or ‐BB and correlated with its low mitogenicity but not with the almost equal induction of the early response genes. It is discussed that no or only very low receptor phosphorylation is required to link receptor activation to the induction of c‐fos, egr‐1 or c‐myc. Furthermore the induction of the investigated gene does not seem to be sufficient for an optimal mitogenic response.

Topological studies suggest that the pathway of the protons through F0 is provided by amino acid residues accessible from the lipid phase

The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. coli F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeled amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologous proteins suggested the existence of tightly packed alpha-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling pattern was observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membranes were pretreated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu-65 which binds DCCD covalently, indicating the location of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit alpha or beta and thus enables proton translocation. Conserved residues in subunit alpha, probably located in the lipid bilayer, might participate in the proton translocation mechanism.

Effect of growth factors on the activation of human Tenon’s capsule fibroblasts

Purpose. To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1ß, TGF-ß1 and TGF-ß2 on the proliferation and myofibroblast transformation of cultured human Tenon’s capsule fibroblasts and to characterize expression of PDGF- and TGF-ß-receptors in these cells. Methods. To determine cell proliferation, cell number of 2nd passage cultured human Tenon’s capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate a-smooth-muscle actin (a-SMA) expression. Expression of PDGF- and TGF-ß-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. Results. A significant increase in proliferation (p = 0.05) was detected after exogenous stimulation with PDGF-AA (10ng/ml and 100ng/ml), PDGF-AB (10ng/ml and 100ng/ml), PDGF-BB (10ng/ml and 100ng/ml), bFGF (100 ng/ml), IL-1ß (1 ng/ml and 10 ng/ml), TGF-ß1 (0.5 ng/ml) and TGF-ß2 (0.5 ng/ml). Both TGF-ß1 and TGF-ß2 stimulated expression of a-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-ß1) and 500 ng/ml (TGF-ß2). Protein and mRNA of PDGF-receptor type a and type ß and TGF-ß-receptors type I, II and III are expressed in cultured human Tenon’s capsule fibroblasts. Conclusions. The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon’s capsule fibroblasts after glaucoma filtering surgery while TGF-ß-isoforms are essential for the transformation of Tenon’s capsule fibroblasts into myofibroblasts.