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Biomedical subjects

G Werner

Publications and source records attributed to G Werner.

At least 37 records · Page 2Linked to original sources

Morphological characterization of the seminiferous cycle in the goat (Capra hircus): a histological and ultrastructural study.

The cycle of spermatogenesis/seminiferous cycle was investigated in the goat testis using both light and electron microscopy techniques. Using the various cell associations and the accompanying changes in spermatid shape and location, the cycle was divided into eight (8) successive stages. The cycle began with the accomplishment of spermiation (stage 1) and ended with apical migration and close attachment of late maturation phase spermatids at the Sertoli cell apex accompanied by adluminal retention of residual bodies with dense staining inclusions (stage 8). The early stages of the cycle (stages 1-4) were therefore characterized by the presence of only one generation of spermatids, the second one appearing only after the division of secondary spermatocytes in stage 4. Consequently, stages 5-8 had two generations of spermatids; Golgi or cap phase as well as maturation phase spermatids. Although stages 5 to 7 appeared as distinct entities, stages 6 and 7 were rather short-lived and considered as continuations of stage 5. Therefore, the 8 stages of the cycle in the goat were further condensed into 6 main divisions. The duration of each stage was estimated by the frequency of occurrence in sections. Among these, stage 1 had the highest frequency (34%) followed by stages 5-7 (27%). Stages 8 and 4 had the shortest frequency (up to 9%) while stages 2 and 3 had 13% and 12% respectively. These results indicate that, like most domestic species, goats have a cycle of 8 stages with 6 main divisions, the longest being stage 1.

Acrosome↗

Linkage of determinants for streptogramin A, macrolide-lincosamide-streptogramin B, and chloramphenicol resistance on a conjugative plasmid in Enterococcus faecium and dissemination of this cluster among streptogramin-resistant enterococci.

A new streptogramin A resistance gene, satG (= vatE), has been recently identified in Enterococcus faecium UW1965 (Werner and Witte 1999. Antimicrob. Agents Chemother. 43: 1813-1814). Further sequence analysis of this plasmid revealed that vatE is in a cluster together with other resistance genes. The identified ORFs were nearly identical with the already known genes ermB and cat. The ermB fragment exhibited more than 99% identity with a resistance region from the streptococcal plasmid pIP501, whereas the cat fragment also contained a truncated rep gene homologue with more than 99% identity to sequences in small staphylococcal plasmids. The cat-rep and the ermB-vatE segments were linked by an IS1216V insertion sequence widely distributed among enterococci. PCR analysis of additional 76 streptogramin-resistant isolates possessing vatE and ermB revealed a linkage of both genes in 45 isolates (59%); 15 of them with a gene arrangement, cat-repU-IS1216V-ermB-vatE, identical to the reference strain UW1965. An identical linkage of IS1216V-ermB-vatE was found among isolates from poultry manure, poultry meat, stool samples of humans, and hospital patients indicating a possible spread of the resistance gene cluster via the food chain to humans.

Anti-Bacterial Agents↗

Short cut of protein purification by integration of cell-disrupture and affinity extraction.

Screening strategies based on functional genomics require the isolation of gene products of several hundred cDNA clones in a fast and versatile manner. Conventional purification strategies will fail to accomplish this goal within a reasonable time frame. In order to short-cut these procedures, we have developed a combination of cell disintegration and affinity technique for rapid isolation and purification. For our purpose, tagged proteins have been produced in yeast by fusing the FLAG-sequence adjacent to the 5' end of cDNAs coding for the respective protein. The example of an over-expressed FLAG-tagged fusion protein, human serum albumin (HSA), was released into the cytoplasm. Detection and purification of the FLAG-fusion protein were carried out by using a mouse monoclonal antibody directed against the FLAG-peptide. For purification purposes, the antibody was immobilized on PROSEP magnetic glass beads. These magnetic glass beads with 500 microns diameter have been investigated for disintegration of yeast and simultaneous capturing of the target protein. After 60 s, 90% of the maximal disintegration level was achieved when a ratio of 20 microliters yeast cell suspension and 100 microliters glass are vortexed. After a wash step, the FLAG-fusion proteins have been eluted with chelating agents such as EDTA. The short-cut procedure has been compared to a conventional purification strategy using an affinity chromatography process. Due to the highly favorable binding characteristics of the applied immunoaffinity sorbent the yield observed in batch operation was 90% and purity in the range of 70-80%.

Amino Acid Sequence↗

Quinupristin/dalfopristin-resistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany.

The semisynthetic streptogramin combination quinupristin/dalfopristin (Synercid) is a promising alternative for treatment of infections due to multiply resistant gram-positive bacteria including vancomycin-resistant Enterococcus faecium. Resistance is mediated by acetyltransferases SatA (VatD) or SatG (VatE). Recent papers have indicated a possible link between the use of the streptogramin virginiamycin S/M as a feed additive in commercial animal husbandry and a selection of quinupristin/dalfopristin-resistant E. faecium (QDRE). We screened manure samples from two different turkey farms and from six different pig farms (using virginiamycin), samples from a sewage water treatment plant, 24 broiler carcasses, 10 pork samples, and 200 stool samples of nonhospitalized humans for QDRE. Our strain culture collection of hospital E. faecium isolates from the last 2 years was also reviewed for QDRE. All manure and sewage samples were positive for QDRE, as well as 11 from broiler carcasses (46%), 1 from pork (10%), and 28 from human stool specimens (14%). Thirty-six hospital isolates of E. faecium exhibited resistance to quinupristin/dalfopristin. In 141 QDRE of different origin satA (vatD) and satG (vatE) genes were detected (seven isolates from humans with an unknown resistance mechanism). Streptogramin resistance determinants were tansferable in filtermating experiments for 5 of 10 satA (vatD) and 9 of 22 satG (vatE) isolates. Different EcoRI patterns of satG (vatE) plasmids and corresponding hybridizations of the satG (vatE) gene indicated nonhomologous resistance plasmids in isolates of different origin. The results of this study indicate a common gene pool for streptogramin resistance in E. faecium of different ecological origin. A selection of QDRE using the streptogramin virginiamycin S/M as a feed additive and a spread of the resistance via the food chain to humans is probable.

Acetyltransferases↗

Impact of flavophospholipol and vancomycin on conjugational transfer of vancomycin resistance plasmids.

The influence of vancomycin and flavophospholipol (FPL) on the transfer rate of conjugative plasmids harboring the vancomycin resistance operon vanA was determined in several clinical and animal isolates of Enterococcus faecium. FPL significantly inhibited the frequency of transfer of conjugative VanA plasmids up to 70-fold. Vancomycin had no significant effect on the transfer rate of VanA plasmids.

Anti-Bacterial Agents↗

Protein expression strategies for identification of novel target proteins.

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.

Blotting, Western↗

Spermatogenesis in the grasscutter, Thryonomis swinderianus (Rodentia).

Spermatogenesis in the grasscutter, Thryonomis swinderianus, is characterized by remarkable diversification of the endoplasmic reticulum. In spermatocytes, besides the usual form with flat cisternae and vesicles, this membrane system forms unusually wide cisternae and vacuoles containing moderately electron dense material near the Golgi apparatus and the cell membrane. In spermatids a whorl of smooth cisternae enclosing aggregates of granular electron dense material appears as another specialization. Although all parts of the endoplasmic reticulum are in connection with each other, their distinct structural differences suggest functionally different subcompartments. Once established, the structures persist even after cytoplasm has been discarded from the nearly mature sperm. In the elongating spermatid a thin plica of the Sertoli cell begins to separate the developing structures from the remainder of the cytoplasm containing the endomembrane systems, part of which is further ensheathed when this fold enroles to form an incomplete tube. Occasionally, in late spermatids a spindle-shaped body of paracrystalline structure occurs temporarily around the flagellum at the beginning of the main piece.

Acrosome↗

Selective pressure by antibiotics as feed additives.

Antibacterial substances are used in considerable amounts as growth promoters in animal husbandry. There are, however, incalculable risks for human health resulting from the use of particular feed additives. Even 30 years ago the detection of transferable antibiotic resistance in Enterobacteriaceae led to the demand that antibiotics used in human chemotherapy, or for which cross-resistance against human therapeutics has been demonstrated, should be prohibited as growth promoters. The application of molecular methods to typing and characterization of bacteria and their resistance genes has provided more concise evidence for the transfer of antibiotic resistance among animal husbandry and humans as to resistance to glycopeptides (vanA gene cluster) and to streptogramins (satA).

Animal Feed↗

Survey of antibiotic resistance among enterococci in North Rhine-Westphalia, Germany.

A surveillance study on antibiotic resistance of enterococcal isolates (n = 730) was carried out in North Rhine-Westphalia, Germany, in 1997. Resistance rates to ampicillin (7.4%), high-level gentamicin (15.0%), high-level streptomycin (27.9%), ciprofloxacin (37.9%), vancomycin (1.5%), and teicoplanin (1.5%) were determined. All vancomycin-resistant enterococci (VRE) carried the vanA gene. SmaI and ApaI macrorestriction patterns indicated an intra- and interhospital spread of VRE.

Bacterial Proteins↗

Inhibition of Fc epsilon RI-mediated activation of mast cells by 2,3,4-trihydropyrimidino[2,1-a]isoquinolines.

Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter gene assay targeting mast cell activation of IgE plus antigen, established in an attempt to identify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). The assay is based on a murine mast cell line designated CPII, stimulation by IgE plus antigen, and a reporter gene construct with the TNF alpha promoter linked to luciferase as a read-out system. Via screening about 50,000 substances, compound 2 was found to inhibit the reporter gene induction in the submicromolar range in this assay. Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthesized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butylithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about 50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 microM and a TC50 of 2.7 microM. In a first profiling in secondary assays, it effectively interfered with the production of mediators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical type I reaction) is inhibited to more than 90% by compound 41, when administered intradermally 90 min before challenge.

Animals↗

Portosystemic hepatic encephalopathy after transjugular intrahepatic portosystemic shunt in patients with cirrhosis: clinical, laboratory, psychometric, and electroencephalographic investigations.

A prospective study of hepatic encephalopathy (HE) including neuropsychiatric and psychometric evaluation, electroencephalography, and determination of arterial ammonia levels was performed in 55 cirrhotic patients treated consecutively by transjugular intrahepatic portosystemic shunt (TIPS). The cumulative HE rate increased from 23.6% within the 3-month interval before TIPS to 50. 9% within the first 3-month interval post-TIPS (P = .003). Significant and independent predictors of HE post-TIPS were the presence of HE pre-TIPS and reduced liver function. The cumulative HE rate declined in the second 3-month interval post-TIPS and reached the pre-TIPS level. Chronic forms of HE exceeding grade I were not observed. In a subgroup of 22 nonencephalopathic TIPS patients, the prevalence of subclinical HE did not change after TIPS. Among individual psychometric tests, the block design test gave the highest proportion of pathological results (about 50%), whereas selective reminding gave the lowest (10%-25%). Electroencephalography (EEG) showed a temporary increase of pathological results at 1 month after TIPS, when patients with overt HE (grade I) were included (proportion of 21.1% before vs. 57.1%, P = .005). Arterial ammonia concentration increased from a mean of 94 +/- 26 microgram/dL to 140 +/- 28 microgram/dL at 3 months after TIPS (P < .001). Elevated ammonia levels persisted. TIPS led to a temporary increase of HE incidence within 3 months. The decline of the HE rate beyond 3 months despite a sustained increase of arterial ammonia levels could not entirely be explained by reduction of shunt flow, nor by alteration of liver function. Instead, cerebral adaptation to gut-derived neurotoxins might be anticipated.

Adolescent↗

[Doppler sonographic short- and long-term studies of portal hemodynamics following transjugular intrahepatic portasystemic shunt (TIPSS)].

Shunt insufficiency due to shunt occlusion or stenosis is frequent after TIPS (about 50% after one year). Controversially discussed is whether Doppler sonography is effective in detecting shunt stenosis or whether regular angiographies are required. The experience with a noninvasive method of surveillance primarily based on Doppler sonography is reported here. 58 patients (35 men, 23 women, mean age 55 years, range 33-82 years) were treated by TIPS because of complications of portal hypertension (43 x gastroesophageal bleeding, 14 x refractory ascites, 1 x venoocclusive disease). Liver cirrhosis (alcoholtoxic etiology in 63%) was present in 55 cases, according to Child-Pugh's classification 23 patients = A, 19 patients = B and 13 patients = C. Within a mean observation period of 14 months, Doppler sonography was performed in three months intervals, endoscopy in six months intervals and angiography only when shunt insufficiency was suspected by Doppler sonography and/or because of clinical events, e.g. recurrent bleeding. Immediately after TIPS, maximal flow velocity and flow volume in the portal vein increased by 116% and 115%, respectively. Three months later, a significant increase of portal vein diameter of about 15% was measured. Shunt flow was initially 2.700 ml/min (one week after TIPS) and decreased progressively by about 30% within the first nine months of follow-up. Correspondingly, angiographically proven shunt insufficiency was present in 22 patients (33 episodes). A total of twelve bleeding episodes recurred in seven patients (rebleeding rate of 16% after one year and 19% after two years). In the remaining 15 patients (68%; 21 episodes) shunt insufficiency could be corrected prior to complications because of detection by Doppler sonography (19x) and endoscopy (2x). Therefore, Doppler sonography is an effective diagnostic tool for the detection of shunt insufficiency and should be performed at three months intervals for at least 18 months. In this context it appears allowable to avoid routine angiographies.

Adult↗

Interstitial tissue in the testis of the giant rat, Cricetomys gambianus.

Besides Leydig cells macrophages form considerable part of the interstitial tissue in the giant rat, Cricetomys gambianus. Leydig cells contain bundles of usually six to twelve parallel tubes with walls of longitudinal layers alternating with circular layers of microfilaments. Polyribosomes, both free and membrane bound, are concentrated around or inside the tubes. The tubes may enclose mitochondria and microperoxisomes as well. The light cytosol of the macrophages is dotted with electron-dense 5-nm particles which might be ferritin. Specific contacts exist between Leydig cells and macrophages indicating a functional interdependence between these two cell types.

Animals↗

[Appendicitis admitting diagnosis. A 3-year analysis].

During a period of three years 863 patients were stationary admitted with a appendicitis as assignment diagnosis. Appendicitis of a disease of the appendix was found in 59.4% of the patients. 40.6% of all admitted patients showed no evidence of appendicitis. Out of these cases, 281 patients (32.6%) were treated conservatively, 69 patients (8%) underwent an operation. The result of the analysis indicated, that the assignment diagnosis appendicitis--now as before--is significantly doubtful having an impact on the subsequential decisions.

Adolescent↗