Real-time quantitative RT-PCR after laser-assisted cell picking.

The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.

Relevance of microbial coculture fermentations in biotechnology.

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro-organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.

[Dysphagia, heartburn and esophageal bolus impaction in a 43-year old male patient: not always reflux disease]. / Dysphagie, Sodbrennen und Bolusobstruktion bei einem 43-jährigen Mann: Nicht immer Refluxkrankheit.

We report a case of a 43 year old male patient, who was admitted with recurring esophageal bolus impactions. Since his childhood he has been complaining about dysphagia and was unable to swallow medication. He also complained about heartburn. The last esophageal bolus impaction was some weeks ago. After elimination of the bolus impaction with a rigid endoscope we found a high grade stenosis in the proximal esophagus that could not even be passed with a children's endoscope. An initial treatment of eosinophil esophagitis would be the therapy with a local corticoid for 6-9 month. In patients with typical rings or stenosis a dilation therapy might be necessary.

[Erionite-induced pleural plaques. Exposition to urban pollution in a female Turkish migrant in Germany]. / Erionitinduzierte Pleuraplaques. Urbane Belastung bei einer türkischen Migrantin.

Erionite is a zeolite mineral of volcanic origin which contains no asbestos. It is found in different regions of the world, including southeast Turkey in ash and dust, from which it can cause inflammatory pleural plaques or malignant pleural mesothelioma (MPM). We report a female Turkish migrant exposed to urban pollution in her home country who decades later suffered from pleural plaques with a nonspecific chronic inflammatory disease. The differential diagnosis of inflammatory pleural plaques was assumed radiologically and confirmed by video-assisted thoracoscopic biopsy. Short-term clinical and radiologic control of the patient will be necessary because of the risk of MPM. For epidemiologic considerations discussed referring to current literature, a growing incidence of this type of disease in migrants from high-risk areas must be reckoned with in Germany, even without exposition to asbestos.

Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae.

Glycerol is the main compatible solute in Saccharomyces cerevisiae. It is accumulated intracellularly when cells are exposed to decreased extracellular water activity. In general, increased intracellular accumulation of a solute may be caused by enhanced production, restricted dissimilation, increased retention by the plasma membrane and increased uptake from the medium. In this review, we evaluate current knowledge concerning mechanisms leading to the accumulation of glycerol in osmotically stressed cells of S. cerevisiae at the molecular and metabolic levels. An overview of glycerol metabolism in S. cerevisiae is provided.

Physiology and genetics of methylotrophic bacteria.

Methylotrophic bacteria comprise a broad range of obligate aerobic microorganisms, which are able to proliferate on (a number of) compounds lacking carbon-carbon bonds. This contribution will essentially be limited to those organisms that are able to utilize methanol and will cover the physiological, biochemical and genetic aspects of this still diverse group of organisms. In recent years much progress has been made in the biochemical and genetic characterization of pathways and the knowledge of specific reactions involved in methanol catabolism. Only a few of the genetic loci hitherto found have been matched by biochemical experiments through the isolation or demonstration of specific gene products. Conversely, several factors have been identified by biochemical means and were shown to be involved in the methanol dehydrogenase reaction or subsequent electron transfer. For the majority of these components, their genetic loci are unknown. A comprehensive treatise on the regulation and molecular mechanism of methanol oxidation is therefore presented, followed by the data that have become available through the use of genetic analysis. The assemblage of methanol dehydrogenase enzyme, the role of pyrrolo-quinoline quinone, the involvement of accessory factors, the evident translocation of all these components to the periplasm and the dedicated link to the electron transport chain are now accepted and well studied phenomena in a few selected facultative methylotrophs. Metabolic regulation of gene expression, efficiency of energy conservation and the question whether universal rules apply to methylotrophs in general, have so far been given less attention. In order to expand these studies to less well known methylotrophic species initial results concerning such area as genetic mapping, the molecular characterization of specific genes and extrachromosomal genetics will also pass in review.

Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts.

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.

Self-splicing of a Podospora anserina group IIA intron in vitro. Effects of 3'-terminal intron alterations on cleavage at the 5' and 3' splice site.

A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI I1) of the ascomycete Podospora anserina can undergo self-splicing in vitro. When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, bI1) the autocatalytic reaction shows a lower efficiency and 5' cleavage takes place predominantly by hydrolysis. In order to test the influence on reaction efficiency and mode of 5' cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI. Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5' transesterification and influences the efficiency of the second splicing step; (2) 5' transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns. Moreover, our data indicate that the postulated gamma-gamma' tertiary interaction is also functional for group IIA introns. A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3' splice site and to a selection of cryptic sites downstream in the 3' exon that almost exclusively restore the strong wild-type gamma-gamma' pairing. The so-called "guide" interaction seems to support the selection of 3' cleavage sites but is of secondary importance in relation to the gamma-gamma' interaction.

Mutant alleles of the MRS2 gene of yeast nuclear DNA suppress mutations in the catalytic core of a mitochondrial group II intron.

Previous studies show that some yeast strains carrying point mutations of domain 5 that block splicing of a mitochondrial group II intron yield spontaneous revertants in which splicing is partially restored by dominant mutations of nuclear genes. Here we cloned and sequenced the suppressor allele of one such gene, and found it to be a missense mutation of the MRS2 gene (MRS2-L232F). The MRS2 gene was first implicated in group II intron splicing by the finding that overexpression of the wild-type gene weakly suppresses the splicing defect of a mutation of another intron. Tetrad analysis showed that independently isolated suppressors of two other domain 5 mutations are also allelles of the MRS2 gene and DNA sequencing identified a new missense mutation in each strain (MRS2-T230I and MRS2-L213M). All three suppressor mutations cause a temperature-sensitive respiration defect that is dominant negative in heterozygous diploids, but those strains splice the mutant intron at the elevated temperature. The three mutations are in a domain of the protein that is likely to be a helix-turn-helix region, so that effects of the mutations on protein-protein interactions may contribute to these phenotypes. These mutations suppress the splicing defect of many, but not all, of the available splicing defective mutations of aI5gamma, including mutations of several intron domains. Protein and RNA blot experiments show that the level of the protein encoded by the MRS2 gene, but not the mRNA, is elevated by these mutations. Interestingly, overexpression of the wild-type protein restores much lower levels of splicing than were obtained with similar elevated levels of the mutated Mrs2 proteins. The splicing phenotypes of these strains suggest a direct role for Mrs2 protein on group II intron splicing, but an indirect effect is not yet ruled out.

Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization.

Characterization of the gene encoding alpha-sarcin, a ribosome-inactivating protein secreted by Aspergillus giganteus.

The filamentous fungus, Aspergillus giganteus, produces the extracellular ribosome-inactivating protein, alpha-sarcin (Sar). The structural gene (sar) encoding Sar was isolated and characterized by sequence analysis and expression in Aspergillus niger. It codes for a precursor of 177 amino acids containing a secretion signal sequence that is absent in the mature protein. The nucleotide sequence contains several typical features of fungal genes, including a short intron of 65 bp. The transcriptional and translational processing signals of the gene are functional in A. niger, but the yield of recombinant protein is low in comparison with the natural producer. A comparison of sar with the gene encoding restrictocin from Aspergillus restrictus revealed a high degree of similarity between both genes. The conservation of the aa sequence suggests that Sar-like proteins may confer a selective advantage to these fungi under certain environmental conditions.

Oxidative stress and ageing in the fungus Podospora anserina.

The ageing phenomenon exhibited by the ascomycetous fungus Podospora anserina can be either delayed or induced by either different carbon sources or effectors. As these effects seem to have analogy to catabolite-repression of respiratory genes, experiments concerning respiratory functions have been carried out. Ageing is parallelled by switching from cytochrome c-oxidase-mediated respiration to alternative, cyanide-resistant respiration for reasons still unknown. The latter is always accompanied by appearance of the phenol oxidizing enzyme laccase (EC 1.10.3.2), which seems to act as an alternative oxidase. The existence of a second, non-mitochondrially encoded respiratory pathway relieves the selective pressure on mitochondria leading to disintegrated, non-functional mtDNA and thereby whole mitochondria which accumulate in the hyphal cells. Mutants lacking cytochrome c-oxidase aa3 or laccase have stable mitochondrial populations and live eternally.

Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking.

Analysis of gene expression and its transcriptional regulation requires a reliable access to target mRNA. However, mRNA extractions from homogenized tissue are limited because only average data are obtained, and cell-specific expression may not be addressed. Here, we describe a new method that combines the microscopic selection of oligocellular clusters or a few isotypic cell profiles from complex tissues by UV-laser-assisted cell picking with a simplified and highly efficient protocol for mRNA amplification. For positive control and quantitation reference, a reliable housekeeping gene is needed. For this purpose, we designed primers of the rat porphobilinogen deaminase (PBGD) gene. In contrast to many commonly used housekeeping primer pairs that co-amplify processed pseudogenes, this sequence allowed detection of a pseudogene-free rat cDNA sequence, thus eliminating the need for a DNase-digestion step. PBGD mRNA was consistently expressed in all complex tissues investigated and in 21 specific cell types harvested by laser-assisted cell picking. PBGD is suggested as a reliable new rat housekeeping gene, particularly suitable for analysis of oligocellular samples such as those obtained by laser-assisted cell picking in complex tissues.

Human transferrin receptor is active and plasma membrane-targeted in yeast.

The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae. The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation. The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells. The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay.

The replication origin of the Methylomonas clara plasmid pBE-2.

The Methylomonas clara narrow host range plasmids pBE-2 and pBE-3 belong to the class of plasmids encoding a trans acting replication initiation factor. Characteristically for such plasmids, the sequence of the origin of pBE-2 and pBE-3 contains a number of large direct repeats (8 and a half iterons of 19 bp), which by analogy are putative binding sites of the trans acting replication factor. Several additional features typical for the majority of E. coli plasmids were found in the M. clara origin: These include sequences homologous to the E. coli DnaA-box, sequences resembling E. coli IHF binding-sites, an AT-rich region with short repeats (similar to those repeats of E. coli origins responsible for an initial DNA duplex opening), and an AT-rich bent DNA region containing inverted repeats which have homology to small repeated sequences found in several plasmid origins. In addition, in the M. clara plasmid origin, large potential hairpin structures are present and the sequence of one of these participates in site specific recombination.

[99mTc-MIBI scintigraphy of hypofunctional thyroid nodules. Comparison of planar and SPECT imaging]. / 99mTc-MIBI-Szintigraphie hypofunktioneller Schilddrüsenknoten. Vergleich von planarer und tomographischer Technik.

UNLABELLED: 99mTc-MIBI-scintigraphy allows to assess the dignity of hypofunctional thyroid nodules. A concordant pattern in MIBI- and pertechnetate-scintigraphy excludes malignancy with high accuracy. For increased MIBI-uptake histological evaluation is advised. The assessment of MIBI-isointense nodules is discussed controversially. Objective of our study was to analyse the prevalence of malignancy for isointense nodules and the diagnostic accuracy of image acquisition in SPECT-technique. PATIENTS, METHODS: MIBI-scintigraphies were analysed retrospectively. Imaging was performed 60 min after intravenous injection of 510 MBq 99mTc-MIBI. Thyroid nodules were assessed as hypo-, iso- or hyperintense compared to the paranodular tissue. RESULTS: 83 of 225 patients underwent thyroid surgery (age 48.6 ± 12.6 years, 72% women). In 12 (14.5%) cases a papillary carcinoma was diagnosed. In planar imaging 12, 34 and 37, in tomographical imaging 16, 21 and 46 nodules, respectively, were classified as hypo-, iso- oder hyperintense. Among hypo-, iso- and hyperintense nodules in planar imaging 1, 5 and 6 carcinomas were found, resp. In tomographical imaging no, 4 and 8 carcinomas were found, respectively. Classification of iso- and hyperintense nodules as "suspicious for malignancy" showed for planar imaging a sensitivity, specificity, NPV and PPV of 91.7, 15.5, 91.7 and 15.6%, for tomographical imaging of 100, 22.5, 100 and 17.9%, respectively. CONCLUSION: Hypofunctional thyroid nodules with iso- and hyperintense MIBI-Uptake have a comparable prevalence of malignancy. Image acquisition in SPECT-technique results in improved diagnostic sensitivity and negative predictive value.

Spore-forming bacteria and their utilisation as probiotics.

In this review article, the beneficial application of bacterial spore formers as probiotics in the food industry is discussed based on the knowledge gleaned from current publications. The summary of new scientific results provides evidence of the advantages of the utilisation of Bacillus or Clostridium strains in the food industry. Both bacteria are able to produce a very stable duration form: the endospore. Compared to the widely used lactic acid bacteria, bacterial spores offer the advantage of a higher survival rate during the acidic stomach passage and better stability during the processing and storage of the food product. In many food products, germination of the spores does not occur. Hence the product quality of the food is not affected because of their inactive metabolism. Besides the possible utilisation and functional properties, an overview of the fast-developing knowledge about the mechanisms of the beneficial health effects of spore-forming bacteria is provided.

[Posthepatic obstructive jaundice caused by primary extragonadal germ-cell tumor in a patient with glucose-6-phosphatase dehydrogenase deficiency]. / Posthepatische Cholestase infolge primär-extragonadaler Manifestation eines Keimzelltumors bei einem Patienten mit Favismus.

HISTORY AND ADMISSION FINDINGS: In patients with glucose-6-phosphatase dehydrogenase (G6PD) deficiency (favism) jaundice is usually caused by hemolysis due to stress, infection or following the application of drugs. We report on a 74-year-old Italian with known G6PD deficiency complaining of jaundice, weight loss and abdominal pain. Physical examination revealed jaundice of the eyes. Scrotal examination by palpation and ultrasound showed no abnormalities. INVESTIGATIONS: Serum levels of beta-human chorionic gonadotropin and alpha-fetoprotein were within normal limits, total bilirubin was extremely elevated, with predominant direct bilirubin. Abdominal ultrasound showed posthepatic blockage of bile flow with a dilated ductus hepatocholedochus (DHC) in the absence of gallstones. Enlarged, multiple contrast-stained paraaortic and retroperitoneal lymph nodes were detected by endoscopic ultrasound and magnetic resonance imaging. Due to failed endoscopic retrograde cholangiopancreatography, visualization of the biliary tree by percutaneous transhepatic cholangiography (PTC) was performed showing an occlusion of the DHC. THERAPY AND COURSE: After successful stent-implantation by PTC with decompression of the biliary tree, the jaundice disappeared. Computer tomography-guided percutaneous biopsy of a retroperitoneal lymph node was performed for histological evaluation showing a primary extragonadal nonseminomatous germ cell tumor. According to the histology (embryonic carcinoma) and clinical stage of the tumor systemic chemotherapy was initiated including cisplatin, etoposide and ifosfamide. After the first cycle of chemotherapy the patient suffered from pneumonia leading to septic shock. Twenty-seven days after admission, the patient died of multiple organ failure. CONCLUSION: Extragonadal germ-cell tumor presenting as retroperitoneal lymph nodes with obstructive jaundice has to be considered in the differential diagnosis of cholestasis.