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M Schrader

Publications and source records attributed to M Schrader.

At least 19 recordsLinked to original sources
PubMed2000

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.

ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.

Amino Acid Sequence↗
PubMed2000

Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom.

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin alpha(5)beta(1) antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.

Amino Acid Sequence↗
PubMed2000

Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K(+)-translocating KdpFABC complex.

Replacement of glycine residue 232 with aspartate in the KdpA subunit of the K(+)-translocating KdpFABC complex of Escherichia coli leads to a transport complex that has reduced affinity for K(+) and has lost the ability to discriminate Rb(+) ions (, J. Biol. Chem. 270:6678-6685). This glycine residue is the first in a highly conserved GGG motif that was aligned with the GYG sequence of the selectivity filter (P- or H5-loop) of K(+) channels (, Nature. 371:119-122). Investigations with the purified and reconstituted KdpFABC complex using the potential sensitive fluorescent dye DiSC(3)(5) and the "caged-ATP/planar bilayer method" confirm the altered ion specificity observed in uptake measurements with whole cells. In the absence of cations a transient current was observed in the planar bilayer measurements, a phenomenon that was previously observed with the wild-type enzyme and with another kdpA mutant (A:Q116R) and most likely represents the movement of a protein-fixed charge during a conformational transition. After addition of K(+) or Rb(+), a stationary current could be observed, representing the continuous pumping activity of the KdpFABC complex. In addition, DiSC(3)(5) and planar bilayer measurements indicate that the A:G232D Kdp-ATPase also transports Na(+), Li(+), and H(+) with a reduced rate. Similarities to mutations in the GYG motif of K(+) channels are discussed.

Adenosine Triphosphatases↗
PubMed2000

Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients.

OBJECTIVE: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes. DESIGN: Prospective observational study. SETTING: A university hospital. PATIENT(S): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell-only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings. INTERVENTION(S): Thirty-three testicular biopsies. MAIN OUTCOME MEASURE(S): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR. RESULT(S): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell-only syndrome showed either telomerase activity or hTERT, but all of them showed hTR. CONCLUSION(S): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures.

DNA-Binding Proteins↗
PubMed2000

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Adenosine Triphosphate↗
PubMed2000

Social network methods for endemic foci of syphilis: a pilot project.

BACKGROUND: Social network methods have improved our understanding of sexually transmitted disease transmission dynamics, and may be of use in routine field operations for partner notification. GOAL: To augment traditional syphilis-control activities with social network methods in an Atlanta area with high syphilis morbidity. STUDY DESIGN: Disease investigators conducted interviews, used network diagrams to prioritize their work, and relied on network connections for finding hard-to-reach persons. RESULTS: A total of 396 contacts were elicited from 48 infected and 50 uninfected persons. The cumulative prevalence of syphilis was 12.6%, and 24 persons infected with HIV were identified. Network methods disclosed a large, interconnected group (276 persons) characterized by high network centrality and the substantial presence of small, interactive subgroups (microstructures). CONCLUSION: The network approach is a feasible field technique, and can identify core groups involved in the intense transmission of syphilis. The targeted, network-based approach may be useful in attempts to eliminate syphilis.

Adult↗
PubMed2000

The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.

Amino Acid Motifs↗
PubMed2000

Real time imaging reveals a peroxisomal reticulum in living cells.

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

Animals↗
PubMed2000

[Revascularization of the corpora cavernosa in ischemia-induced erectile dysfunction].

Revascularization of the cavernous bodies (CB) has its place in a highly selected patient population as the only causal therapy for erectile dysfunction (ED) and provides an alternative to the implantation of alloplastic erectile aids. The indication for arterialization of the CB is currently only established when conservative treatment has failed. The most important criteria for the intervention are controversial: an age of under 50, an incidence of not more than two classical risk factors for impotence and the exclusion of diabetes mellitus. The leading revascularization procedure in German-speaking countries comprises arteriovenous shunting of the inferior epigastric artery with the dorsal vein and artery of the penis. A retrospective analysis of results is problematic due to the nonuniform indicational criteria, the multiplicity of applied revascularization procedures and a nonstandardized follow-up. Subjective assessment of improved erectility is the sole target criterion in the majority of studies. Therapeutic results range from 33 to 87% with regard to subjective success rates. Attempts to objectify the treatment results were made in only few of the studies and disclosed bypass patency in 44 to 92% one year after the intervention. The conclusions drawn at the last "Consensus Development Conference on Impotence" (CDCI) of the National Institutes of Health (NIH) in Washington have not lost their topicality in view of the great number of still unanswered questions. There the recommendation was made in 1992 to perform penile arterialization only in controlled prospective clinical trials. The European Urological Association (EUA) is currently organizing a Consensus Conference that will provide the framework for prospective studies that can serve as a basis for clarifying the open questions.

Aged↗
PubMed1999

Composition of the peptide fraction in human blood plasma: database of circulating human peptides.

A database was established from human hemofiltrate (HF) that consisted of a mass database and a sequence database, with the aim of analyzing the composition of the peptide fraction in human blood. To establish a mass database, all 480 fractions of a peptide bank generated from HF were analyzed by MALDI-TOF mass spectrometry. Using this method, over 20000 molecular masses representing native, circulating peptides were detected. Estimation of repeatedly detected masses suggests that approximately 5000 different peptides were recorded. More than 95% of the detected masses are smaller than 15000, indicating that HF predominantly contains peptides. The sequence database contains over 340 entries from 75 different protein and peptide precursors. 55% of the entries are fragments from plasma proteins (fibrinogen A 13%, albumin 10%, beta2-microglobulin 8.5%, cystatin C 7%, and fibrinogen B 6%). Seven percent of the entries represent peptide hormones, growth factors and cytokines. Thirty-three percent belong to protein families such as complement factors, enzymes, enzyme inhibitors and transport proteins. Five percent represent novel peptides of which some show homology to known peptide and protein families. The coexistence of processed peptide fragments, biologically active peptides and peptide precursors suggests that HF reflects the peptide composition of plasma. Interestingly, protein modules such as EGF domains (meprin Aalpha-fragments), somatomedin-B domains (vitronectin fragments), thyroglobulin domains (insulin like growth factor-binding proteins), and Kazal-type inhibitor domains were identified. Alignment of sequenced fragments to their precursor proteins and the analysis of their cleavage sites revealed that there are different processing pathways of plasma proteins in vivo.

Amino Acid Sequence↗
PubMed1999

Role of dynactin in endocytic traffic: effects of dynamitin overexpression and colocalization with CLIP-170.

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170's putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

Animals↗
PubMed1999

Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells.

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.

Acetylcysteine↗
PubMed1998

Expression of PEX11beta mediates peroxisome proliferation in the absence of extracellular stimuli.

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.

Amino Acid Sequence↗
PubMed1998

Human beta-defensin-1: A urinary peptide present in variant molecular forms and its putative functional implication.

Human beta-defensin-1 (hBD-1) was first isolated from blood filtrate by our group. Further studies elucidate the significance of this peptide in the human urogenital tract. The hBD-1 gene is expressed in urogenital epithelial organs such as urinary bladder, ureter, vagina and particularly in distal tubular cells of the kidney. Functional characterization of hBD-1 was carried out with native hBD-1 purified from human body fluids. Several different N-terminally truncated variants derived from the 68-amino acid-containing precursor of hBD-1 occur in blood filtrate and in urine. The generation of these variants can be explained by digestion through a chymotrypsin-like protease. Unlike the alpha-defensins which are structurally related peptide antibiotics, our results indicate that native hBD-1 exhibits minor antimicrobial activity which is not related to the extension of the N-terminus. Only few microorganisms, for example bacilli, are significantly inhibited by hBD-1. Moreover, antibiotic activity is suppressed in solutions containing physiological sodium chloride concentrations. This is in contrast to previous reports assuming a pivotal role of hBD-1 in antimicrobial host defense. In contrast to its weak antimicrobial activity, it is shown that hBD-1 has a strong cytotoxic potential towards mammalian cells like NIH-3T3 fibroblasts. We assume that this property might be important during eradicative processes at epithelia in particular when the synthesis rate of this peptide is upregulated.

3T3 Cells↗
PubMed1998

Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.

5'-Nucleotidase↗
PubMed1998

Distinction between the three disulfide isomers of guanylin 99-115 by low-energy collision-induced dissociation.

Guanylin 99-115 is a small human peptide hormone with two disulfide bonds. The four cysteinyl residues in this peptide allow the formation of two disulfide bridges in three different ways but only the 1-3/2-4 combination is able to bind to the receptor and cause an increase of intracellular cGMP, while the other isomers are biologically inactive. Using guanylin 99-115 as a model peptide, the aim of this study was to investigate whether it is possible to distinguish the differently bridged isomers directly by tandem mass spectrometry. Guanylin isomers were generated by performing an air oxidation of fully reduced guanylin 1-3/2-4 obtained by chemical synthesis. The reaction product is a mixture of guanylin 1-4/2-3 and guanylin 1-2/3-4 in a ratio of 3:1, but there is virtually no guanylin 1-3/2-4. The two biologically inactive peptides were separated by reversed-phase high pressure liquid chromatography (HPLC). Using low-energy collision-induced dissociation tandem mass spectrometry, it was possible to distinguish unambiguously between the three guanylin isomers. This was possible due to the identification of a large number of fragments with intact disulfide bonds. Accordingly, this strategy of a direct and sensitive analysis should work as well for other peptides, with the potential to determine an undefined disulfide bond pattern.

Amino Acid Sequence↗