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Biomedical subjects

G Schenk

Publications and source records attributed to G Schenk.

At least 19 recordsLinked to original sources
PubMed2001

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center.

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.

Acid Phosphatase↗
PubMed2001

Gene therapy: future therapy for erectile dysfunction.

Advances in molecular biological techniques, completion of the Human Genome Project, and the ensuing age of molecular medicine, in conjuction with the sum of a decades-long accumulation of knowledge of the physiology of erection and the pathophysiology of erectile dysfunction have converged to make gene therapy for erectile dysfunction a distinct possibility. In short, both the intrinsic complexities of mechanisms responsible for ensuring normal erection and the multifactorial nature of erectile dysfunction ensure that there is a relatively vast number of physiologically relevant molecular targets for gene therapy. As such, perhaps it is not surprising that virtually every preclinical gene therapy strategy/target examined thus far has been largely successful in ameliorating conditions associated with compromised erectile function in vivo and/or in vitro. This report highlights the goals and strategies of gene therapy for erectile dysfunction and reviews the strategies that initially have been employed. In short, the preclinical data, while still quite preliminary in many regards, are nonetheless quite impressive and encouraging. If similar success is obtained in clinical trials, gene therapy for erectile dysfunction may provide the first concrete "proof of concept" for using gene therapy in the treatment of human smooth muscle disorders.

Erectile Dysfunction↗
PubMed2000

Purple acid phosphatases from bacteria: similarities to mammalian and plant enzymes.

Mammalian and plant purple acid phosphatases have similar active site structures despite low sequence identity (<20%). Although no bacterial enzyme has been purified, a sequence database search revealed that genes that could encode potential purple acid phosphatases may be restricted to a small number of organisms (i.e. myco- and cyanobacteria). Analysis of their deduced amino acid sequences and predicted secondary structures indicates that the cyanobacterial enzyme is similar to both the mammalian and the recently discovered low-molecular-weight plant purple acid phosphatases, while the mycobacterial enzyme is homologous to the fungal and high-molecular-weight plant purple acid phosphatases. Homology models indicate that both bacterial proteins appear to be similar to mammalian purple acid phosphatases in the immediate vicinity of the active site. It is likely that these enzymes act as Fenton-type catalysts in order to prevent damage caused by reactive oxygen species generated by invaded host cells (M. tuberculosis) or by the light-harvesting complex (Synechocystis sp.).

Acid Phosphatase↗
PubMed2000

Identification of mammalian-like purple acid phosphatases in a wide range of plants.

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal approximately 35kDa, plant approximately 55kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.

Acid Phosphatase↗
PubMed2000

Lack of antimicrobial effect on periodontopathic bacteria by ultrasonic and sonic scalers in vitro.

BACKGROUND: The purpose of this study was to assess the antimicrobial effects of a sonic and ultrasonic scaler generally used for subgingival scaling on gram-negative and gram-positive periodontopathic bacteria. METHOD: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, or Peptostreptococcus micros were suspended in Schaedler's broth medium and treated by a sonic or a magnetostrictive ultrasonic scaler for 30 s and 150 s in vitro. Bacterial suspensions treated by an ultrasonic cell disruptor served as a positive control and untreated bacterial suspensions served as a negative control. Following sonication, samples were serially diluted, streaked on blood agar plates and incubated for 2-5 days at 37 degrees C. RESULTS: Treatment by the sonic or ultrasonic scaler for up to 150 s did not reduce the viability of any of the tested periodontal pathogens. Compared to untreated controls, the viability of A. actinomycetemcomitans and P. gingivalis was significantly (p<0.05) reduced only following ultrasonication with the cell disruptor after 30 s (0.72 and 0.54 log CFU/ml, respectively) and of A. actinomycetemcomitans, P. gingivalis, C. rectus, and P. micros after 150 s (1.98, 1.34, 1.95 and 1.98 log CFU/ml, respectively). CONCLUSION: The data of the study may indicate that the assessed sonic and ultrasonic scaler used for subgingival debridement do not result in killing of the tested periodontal pathogens.

Colony Count, Microbial↗
PubMed1999

Binuclear metal centers in plant purple acid phosphatases: Fe-Mn in sweet potato and Fe-Zn in soybean.

Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII-ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6-0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8-0.9 g-atom of iron, 0.7-0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550-560 nm, with molar absorption coefficients of 3200 and 3300 M(-1) cm(-1), respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. The sweet potato enzyme is the first well-defined example of an Fe-Mn binuclear center in a protein.

Acid Phosphatase↗
PubMed1999

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato.

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe-Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe-Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A and have the symmetry of space group P6(5)22, with one dimer per asymmetric unit. Diffraction data collected using a conventional X--ray source from a cryocooled crystal extend to 2.90 A resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Acid Phosphatase↗
PubMed1998

Heterologous expression of human transketolase.

Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Purification of recombinant transketolase and separation from the E. coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag. The highest specific activity was 13.5 U/mg and the K(m) values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K(m) values for thiamin diphosphate and for Mg2+ were, respectively, 4.1 +/- 0.8 and 2.5 +/- 0.4 microM, but after 1 h of preincubation these values were 85 +/- 16 nM and 0.74 +/- 0.23 microM. The kinetic constants are similar to those of the native enzyme purified from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.

Cloning, Molecular↗
PubMed1998

Properties and functions of the thiamin diphosphate dependent enzyme transketolase.

This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall flux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modification of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals.

Amino Acid Sequence↗
PubMed1997

The role of His113 and His114 in pyruvate decarboxylase from Zymomonas mobilis.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zymomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and His114) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gln does not have a great effect on cofactor and substrate binding. However, the k(cat) is decreased indicating that His114 may assist in catalysis. In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Gln113]PDC mutant is capable of releasing acetaldehyde from 2-(1-hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105-112) that precedes His113 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.

Acetaldehyde↗
PubMed1997

Molecular evolutionary analysis of the thiamine-diphosphate-dependent enzyme, transketolase.

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.

Amino Acid Sequence↗
PubMed1997

Controlled local delivery of tetracycline HCl in the treatment of periimplant mucosal hyperplasia and mucositis. A controlled case series.

The purpose of this controlled case series was to assess the adjunctive efficacy of controlled topical tetracycline HCl application in the treatment of infection associated periimplant mucositis or mucosal hyperplasia. Eight patients with at least 2 endosseous implants showing clinical signs of periimplant mucosal hyperplasia or mucositis were enrolled. All implants received supra- and subgingival scaling, with half of the implants receiving adjunctive controlled local delivery of tetracycline HCl (test). Control implants did not receive any other therapy aside from scaling. Clinical parameters were assessed at baseline, 4, and 12 weeks. Scaling plus controlled local delivery of tetracycline HCl markedly reduced periimplant mucosal hyperplasia in 4 of 5 test implants and demonstrated a trend towards a reduction of bleeding on probing scores. Scaling alone had no effect on mucosal hyperplasia in the 2 control implants presenting with this condition nor bleeding on probing scores. In both groups, plaque index scores were slightly reduced at 4 weeks but returned to baseline values at 12 weeks, whereas pocket probing depths, clinical attachment levels, and probing bone levels remained unchanged during the course of the trial. The observed trends suggest that scaling plus controlled local delivery of tetracycline HCl may have beneficial effects. Randomized controlled trials employing a sample size high enough to reach sufficient statistical power are needed to definitively assess the efficacy of controlled local tetracycline HCl delivery on periimplant diseases.

Administration, Topical↗
PubMed1996

Experiences of photodynamic therapy in dermatology.

From March 1988 to May 1994, 15 patients underwent the treatment protocol of superficial photodynamic therapy (PDT) in dermatological localized malignancies. Two tumours (one M. Queyrat of the penis, one basalioma) were treated primarily; the other 13 patients experienced relapses of underlying disease after treatment by surgery, radiotherapy or chemotherapy. Most of the patients (10/15) received doses of 2 mg Photosan III kg-1 body weight (BW), four received a lower dose of 1-1.5 mg kg-1 BW and one patient received 3 mg kg-1 BW. Only one remarkable side-effect (tachyarrhythmia) during Photosan III infusion occurred. All patients were treated by an argon dye laser system. The light dose was 200 J cm-2 (11 patients) and 150 J cm-2 (four patients). Complete response occurred in six lesions, including one M. Queyrat and five basaliomas. Two patients had significant partial response with distinct regression of tumour (one pretreated basalioma of the upper lip and one superficially spreading mesothelioma of the scrotum). Five patients (all basaliomas) demonstrated only local response and two patients gave no response (one basalioma, one melanoma metastasis). The results obtained from this small sample suggest the necessity of an optical dosimetric system. At present, PDT should be restricted for selected or pretreated cases.

Basal Cell Carcinoma↗
PubMed1995

[ROKO--computer-assisted roentgen quality control].

To guarantee the quality of 30 x-ray machines and 8 photographic processors 24,000 data are collected yearly. A computer system is introduced (ROKO-Röntgenkonstanzprüfung) which helps to collect the monthly data easily and to measure on-line dose and voltage. If given tolerance values are exceeded acoustic and graphic warnings appear, a trend analysis is made to assure the correctness of the collected data, a generator to design the layout of check form facilitates the controlling and an on-line documentation for each x-ray machine is available. All these features help save time and money.

Computer Graphics↗
PubMed1991

Opioid-induced respiratory depression and analgesia may be mediated by different subreceptors.

Use of selective delta opioid antagonists provide evidence that the delta receptor within the brain seems an integrated part in the mediation of respiratory depression induced by a potent analgesic like fentanyl. Low doses of the delta antagonists RX-8008M (3-6 micrograms/kg) as well as ICI 174,864 (3-6 micrograms/kg) reversed fentanyl-related respiratory depression (arterial blood gases) in the unanesthetized canine. Opioid-induced blockade of afferent sensory nerve volleys (amplitude height of the somatosensory-evoked potential) could be reversed only by a high dose (9 micrograms/kg) of RX-8008M. Depression of amplitude height of the SEP could not be reversed by ICI 174,864 over the whole dose range (3-6-9 micrograms/kg). In comparison, naloxone (1-5-10 micrograms/kg) not only reversed depression of PaO2, it also reversed the blockade of afferent sensory nerve impulses in the low (5-micrograms/kg)-dose range. A highly selective delta antagonist may have a therapeutic value in reversing opioid-related respiratory depression, resulting in little or no attenuation of analgesia.

Analgesia↗
PubMed1991

[MRT of orofacial tumors--initial clinical experiences with turbo-flash intensity studies].

We examined 36 patients with carcinomas of the tongue and the floor of the mouth. In 11 cases, a clear definition of the tumor extent was not possible using T2 w. spin echo sequences and T1 w. spin echo and gradient echo sequences after injection of contrast material. This delineation was attained with Turbo-FLASH intensity-vs-time studies (Gad-DTPA). The time of acquisition of each measurement was about 1 sec. At the end of injection of Gad-DTPA, 30 measurements with a delay of 1 sec between two measurements were performed. Using this technique, a marginal zone at the boundary of the tumor of very high signal intensity was visible within first minute after Gadolinium injection. This method is not required in the case of clearly hypointense (majority of the tumors) or hyperintense (minority) lesions on T1 w. Gadolinium-enhanced SE or GE images.

Aged↗