[Fine-needle aspiration cytology of thyroid nodules: molecular diagnostics in a routine diagnostic setting]. / Feinnadelzytologie von Schilddrüsenknoten: Molekulare Diagnostik in der klinischen Routine.

BACKGROUND AND OBJECTIVE: Results for the detection of point mutations and rearrangements have thus far been obtained by fresh material of fine needle aspiration cytology (FNAC). After a first retrospective study we report on the diagnostic detection in routinely obtained, consecutive air-dried FNAC smears. METHODS: RNA and DNA was extracted from 154 consecutive routine air-dried FNAC smears: 80 with microfollicular proliferation (MFP), 45 with follicular neoplasia (FN), 26 with the cytological diagnosis of papillary carcinomas (PTC) and 3 which were suspicious for malignancy. PAX8/PPARG and RET/PTC3 rearrangements were detected by qPCR, while BRAF and RAS point mutations were detected by pyrosequencing. RESULTS: Only 0.7 % and 5.3 % of the routine air-dried FNAC samples did not allow analysis of a point mutation or rearrangements, respectively. NRAS mutations could be detected in 7 MFP smears, and in one of FN and PTC samples, respectively. HRAS mutations were detected in one MPF and one FN sample. A KRAS mutation was only detected in one FN sample, whereas BRAF mutations were detected in 20 samples with PTC (but in no other sample). PAX8/PPARG was detected in 2 MFP samples, while RET/PTC was detected in only one MFP sample. In total, 13.8 % MFP-FNAC, 6.7 % FN-FNAC, and 80.8 % PTC-FNAC samples harbored a mutation. CONCLUSION: These results demonstrate that rearrangements and point mutations can be detected in routinely obtained air-dried FNAC samples.

[Scintigraphically "hot" thyroid nodules mainly go hand in hand with a normal TSH]. / Szintigraphisch mehranreichernde Schilddrüsenknoten gehen überwiegend mit normwertigem TSH einher.

AIM: In recent years, various professional societies published guidelines for diagnostic evaluation of thyroid nodules, in which the indication for scintigraphy is restricted to patients with subnormal TSH values. It is seen controversial whether such recommendations should be transferred to Germany, partly because of lower iodine intake in this country and the consequent higher percentage of autonomous thyroid nodules, which are not accompanied by a measurable dysfunction. Since reliable data to this topic are scarce, we analyzed multicentrically the spectrum of scintigraphically "hot" and "warm" nodules under the current epidemiological conditions. PATIENTS, METHODS: In 10 German nuclear medicine out-patient institutions we evaluated the diagnostic data from a total of 514 patients, in whom unequivocally hyperfunctional nodules (focal increased uptake in comparison to perinodular tissue with a sonographically nodular correlative ≥1 cm) could be detected by (99m)Tc-pertechnetate scintigraphy. To minimize selection bias, the surveys were not carried out in hospitals.The recorded parameters included the thyroid hormone levels, the global (99m)Tc-uptake (TcTU), the size of each nodule and the total autonomous nodular volume (V(aut)). RESULTS: Only 20% of the patients with "hot" nodules had subnormal TSH levels (<0.1 to 0.33 mU / l), the remaining patients had TSH levels from 0.34 to 3.5 mU /l (in one third of the patients TSH levels even exceeded 1.0 mU/l). Moreover, we found no relevant correlation between TSH and TcTU or V(aut). CONCLUSIONS: In Germany, in at far the largest proportion of patients with autonomous thyroid nodules objectified by means of scintigraphy, TSH levels are within the normal range. Since such nodules with maximum safety can be classified as benign, a corresponding scintigraphic finding has a high priority for the patient. These current data support that it is not reasonable to restrict scintigraphy to patients with subnormal TSH values in this country.

The Hsp70-Ydj1 molecular chaperone represses the activity of the heme activator protein Hap1 in the absence of heme.

In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

Functional analysis of heme regulatory elements of the transcriptional activator Hap1.

Heme regulation of the activity of diverse proteins was thought to be mediated by heme-responsive motifs (HRMs). The yeast transcriptional activator Hap1 contains seven HRMs: HRM1-7. Three copies of a 17-amino-acid repeat are also located in the region encompassing HRM1 to -6. We examined the effects of these HRMs and repeats on heme regulation of Hap1 activity by deletion analysis and by Ala substitutions of key residues. We found that the effect of mutation or deletion of one HRM or 17-amino-acid repeat on Hap1 heme responsiveness is different from the effect of mutation or deletion of another HRM or repeat. Our data suggest that HRM7 plays a dominant role in mediating heme activation of Hap1 in heme-sufficient cells while HRM1-6 may scavenge heme and cause a low level of Hap1 activation in heme-deficient cells. These results may help in understanding the roles of HRMs in other hemoproteins.

The coiled coil dimerization element of the yeast transcriptional activator Hap1, a Gal4 family member, is dispensable for DNA binding but differentially affects transcriptional activation.

The heme activator protein Hap1 is a member of the yeast Gal4 family, which consists of transcription factors with a conserved Zn(2)Cys(6) cluster that recognizes a CGG triplet. Many members of the Gal4 family contain a coiled coil dimerization element and bind symmetrically to DNA as homodimers. However, Hap1 possesses two unique properties. First, Hap1 binds asymmetrically to a direct repeat of two CGG triplets. Second, Hap1 binds to two classes of DNA elements, UAS1/CYC1 and UAS/CYC7, and permits differential transcriptional activation at these sites. Here we determined the residues of the Hap1 dimerization domain critical for DNA binding and differential transcriptional activation. We found that the Hap1 dimerization domain is composed of functionally redundant elements that can substitute each other in DNA binding and transcriptional activation. Remarkably, deletion of the coiled coil dimerization element did not severely diminish DNA binding and transcriptional activation at UAS1/CYC1 but completely abolished transcriptional activation at UAS/CYC7. Furthermore, Ala substitutions in the dimerization element selectively diminished transcriptional activation at UAS/CYC7. These results strongly suggest that the coiled coil dimerization element is responsible for differential transcriptional activation at UAS1/CYC1 and UAS/CYC7 and for making contacts with a putative coactivator or part of the transcription machinery.

The yeast heme-responsive transcriptional activator Hap1 is a preexisting dimer in the absence of heme.

In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.

A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator.

Heme is a key molecule in mediating the effects of oxygen on various molecular and cellular processes in many living organisms. In the yeast Saccharomyces cerevisiae, heme serves as a secondary signal for oxygen; intracellular heme synthesis directly correlates with oxygen tension in the environment. In yeast, oxygen sensing and heme signaling are primarily mediated by the heme activator protein Hap1, which, in response to heme, activates the transcription of genes required for respiration and for controlling oxidative damage. Heme regulation of many genes required for anaerobic growth is mediated by the aerobic repressor Rox1, whose expression is controlled by heme. In this review, we summarize recent knowledge about (i) how heme synthesis may be controlled by oxygen tension, (ii) how heme precisely and stringently controls Hap1 activity and (iii) whether other transcriptional activators can also mediate heme action.

Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1.

Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization. To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast heme-responsive transcriptional activator HAP1. HAP1 activity is regulated precisely and tightly by heme. Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex. Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1. The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity. Further, we determined HAP1 domains required for heme regulation: three domains-the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain-cooperate to form the higher-order complex and mediate heme regulation. Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation. Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

Immunocytochemical localization of taurine in the pineal organ and retina of an anadromous fish, Plecoglossus altivelis.

Light and electron microscopic immunolocalization of taurine, a sulfur-containing free amino acid, was investigated in the photoneuroendocrine pineal organ and the retinal photoreceptor and pigment epithelial layer of the ayu Plecoglossus altivelis, an anadromous fish. Intense immunostaining was found in the outer segments of pineal photoreceptors and retinal cone-like cells. Moderate but definite immunostaining was found in the cytoplasmic processes of pineal supporting cells, the outer segments of retinal rod-like cells, and the apical processes of pigment epithelial cells. Although the electron microscopic immunogold labeling was not completely coincident with the results of light microscopic immunostaining, concentrated immunogold particles appeared in the inner segments of photoreceptor cells, the cytoplasmic processes of pineal supporting cells, and the apical processes of pigment epithelial cells. These light and electron microscopic findings in taurine immunolocalization were discussed in relation to the functions of taurine known mainly from retinal physiology. It was suggested that the abundant taurine localization may be involved at least in the protection of photoreceptor outer segments against harmful factors, and in the transportation of nutrients and metabolites. The immunostaining for taurine is useful for the discrimination of different types of photoreceptor cells in the pineal organ and retina of fish.

Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli, such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli. To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.

Immunocytochemical demonstration of taurine in the retina and pineal organ of the pigeon.

Taurine-like immunoreactivity (TLI) is found in the pigeon retina at high levels within cones, in select cells of the inner nuclear layer and in sublaminae of the inner plexiform layer. At the ultrastructural level a high density of immuno-gold particles in visualized in the ellipsoid, but not in the paraboloid area of the inner segment. It is present in perikarya and the presynaptic endings of cones, but not in postsynaptic cell processes. The immuno-gold particles are not associated directly with the synaptic vesicles. In the pineal organ TLI was confined to the so-called dark pinealocytes which are surrounded by only family stained light pinealocytes. Clusters of protein A-gold particles are located in the ergastoplasm between polyribosomes adjacent to fibrous proteins and in chains along filamentous structures. Granules, vesicles and membrane whorls, which originate from modified cilia remain unlabeled. The results are discussed on the basis of previous findings; a direct or indirect functional relationship of taurine to ATP-dependent processes is suggested.

[Radiation exposure from nuclear medicine studies in children]. / Strahlenexposition bei der nuklearmedizinischen Untersuchung von Kindern.

Nuclear medical examinations of children have to be performed with special regard to the problems of radiation protection because of the high radiation sensitivity esp. of infants and young children. The present contribution describes how any unnecessary radiation exposure can be avoided by the correct choice and planning of a nuclear medical study, by using the appropriate radiopharmaceutical as well as by the exact calculation of the amount of activity applied, depending on body surface resp. body weight of the child. A technically optimized method which employs the best technical equipment and personnel, being specially trained for working with children, are important conditions to achieve optimal results of nuclear medical tests. Due to the difficulties of direct dose measurements, large variations in the biokinetic behaviour of radiopharmaceuticals and the restriction to standard phantoms, individual dose calculations or dose estimations in pediatrics cause great problems. This is reflected by often large variations of dosimetrical data given in the literature.

[Nuclear medicine diagnosis and therapy in urology. Diagnosis of bone metastases]. / Nuklearmedizinische Diagnostik und Therapie in der Urologie. Diagnostik von Knochenmetastasen.

Bone scintigraphy with 99mtechnetium-labelled polyphosphonates is the most sensitive test for early detection of skeletal metastases. Bone metastases are a major factor in prognosis and have a considerable influence on the therapy selected. In patients with prostate cancer, we recommend routine bone scintigraphy in the initial staging. Follow-up bone scans are indicated whenever a patient develops pain, an elevated level of acid phosphatase, or a rise in prostate specific antigen (PSA). Routine bone scans are not necessary for the initial staging of patients with renal cell carcinomas, bladder carcinomas and testicular tumours. Scans should be routinely performed, however, in patients with bone pain or elevated alkaline phosphatase or when radiological findings are inconclusive. Bone scanning is necessary in patients with neuroblastoma, both for the initial diagnosis and during follow-up in all cases with known skeletal involvement. In addition, bone scintigraphy should be performed in cases of recurrent or suspected malignant phaeochromocytoma as a complement to scintigraphy with iodine-123- or iodine-131-MIBG, respectively. Even though skeletal scintigraphy is a very sensitive test, it lacks specificity. This can be compensated, however, by careful interpretation of the scan in the light of the patient's history and the clinical findings. As a positive side-effect, bone scanning--especially in the form of multiphase scintigraphy--may detect renal abnormalities, concurrent diseases or complications in the upper or lower urinary tract. If scintigraphic findings are doubtful, plain film radiographs are required or, in selected cases, bone biopsy must be performed.

CEA-immunoscintigraphy with 99m-technetium correlates with tumour cell differentiation in colorectal cancer.

The clinical usefulness of immunoscintigraphy with the monoclonal anti-CEA (carcinoembryonic antigen) antibody BW431/26, directly labelled with 99m-Technetium in targeting colorectal carcinomas was investigated in 43 patients. In addition, tumour cell grading and CEA-expression were examined immunohistochemically. Best imaging results were obtained in pelvic tumour lesions (sensitivity 80%). Tumour grading correlated with radioimmunoimaging, well differentiated tumours being detectable at a higher rate (P = 0.09). Immunoscintigraphy preceded the findings of conventional diagnostic methods in 3 patients. In 4 cases immunoscintigraphy was decisive for patients management. Therefore, immunoscintigraphy with 99m-Technetium is valuable in directing patients management if conventional diagnostic methods remain undecisive.

The value of nuclear medicine for the diagnosis of spine diseases.

Nuclear medicine examinations hold an important position in the diagnosis of diseases of the spine. During the last decade, decisive progress has been made in the field of instrumentation and radiopharmaceutical techniques: the use of high resolution collimators and the introduction of emission computer tomography as examples of improved instrumentation as well as 99m-Technetium red blood cell labelling as a new radiopharmaceutical technique. These present some of the developments responsible for the growing importance of scintigraphical diagnosis. Inflammatory processes of the vertebrae and the surrounding soft tissues can be detected or excluded with high reliability by the use of radionuclide-labelled granulocytes. The important role of bone scintigraphy in the differential diagnosis of neoplastic bone disease relies on its high sensitivity combined with the quantitative analysis of increased bone metabolism. Furthermore, it provides exact information about the extent and a possible metastatic spread of bone tumours. In the field of orthopaedy and surgery, skeletal scintigraphy is of growing importance as a highly sensitive procedure in the detection of special traumatic lesions such as acute vertebral compression fractures and in the follow-up of patients after bone surgical interventions. Despite the progress of other imaging modalities such as computer tomography and magnetic resonance imaging, nuclear medicine today is well-established in the assessment of diseases of the vertebral column. Among all scintigraphical diagnostic procedures, bone scintigraphy and the different techniques of inflammation imaging are of special importance.

[Importance of whole body skeletal scintigraphy within the scope of staging of neoplasms in the ENT area]. / Bedeutung der Ganzkörper-Skelettszintigraphie im Rahmen des Staging von Malignomen im HNO-Bereich.

Concerning malignant tumours of the oral cavity, pharynx and larynx, bone metastases are in general rarely seen. For the specification to which patients the whole body bone scintigraphy as detection method should be applied, the findings of 370 patients were analysed retrospectively. In respect of primary staging, bone metastases could be found by scintigraphy in only 1.4% of the patients. On the other hand, there was a detection rate of 12% during the further course of the disease, especially in case of clinical symptoms pointing at spreading metastases or in tumour recurrences. Nevertheless, positive scan findings which were not due to metastases could be found in both groups with equal frequency (12 and 13%, respectively). Therefore the routine performance of whole body bone scintigraphy as a screening method does not seem to be useful in the primary staging of cancer of the mouth, pharynx and larynx. Contrary to this, in the follow-up of these tumours bone scanning proves to be a valuable and sensitive method for detecting skeletal metastases.

[The clinical relevance of anti-CEA immunoscintigraphy with the 99mTc-labelled monoclonal antibody BW 431/26. A critical assessment after 119 studies]. / Klinische Relevanz der Anti-CEA-Immunszintigraphie mit dem 99mTc-markierten monoklonalen Antikörper BW 431/26. Kritische Bestandsaufnahme nach 119 Untersuchungen.

The results of 119 radioimmunoscintigraphies (RIS) in 113 patients with the 99mTc-labeled monoclonal anti-CEA-antibody BW 431/26 (Behring) have been analysed. The aim of our study was the estimation of the method's sensitivity and specificity under different aspects to find out for which indications and questions the 99mTc-RIS is useful. Colorectal primary tumours in 19 patients were scintigraphically detected with a sensitivity of 83% and a specificity of 100%; 3 out of 7 other tumour sites were localised correctly. 55 patients were examined during the follow-up of colorectal cancer. There were 17 out of 22 true positive findings of local recurrences (sensitivity 77%, specificity 88%). Liver metastases were imaged as hot lesions with only 41% sensitivity and 86% specificity. The detection of extrahepatic tumour sites is difficult because of the persistently high blood-pool activity of the monoclonal antibody and, in the pelvic area, the unspecific bowel activity. Skeletal metastases were recognised in 7 out of 9 cases. In 14 patients with other non-colorectal carcinomas, RIS was successful in single cases. It is not helpful, however, when searching for tumours of unknown origin or for the screening of patients with elevated CEA levels without tumour history. The high technical, methodological and time effort required by RIS is justified in the follow-up of cancer patients when conventional diagnostic procedures are inconclusive or the status of morphological findings remains unclear. The use of RIS as an unspecific screening tool in tumour diagnosis must be rejected because of the not completely explored risks of the examination. Repeated applications of monoclonal antibodies require controls of the patients' HAMA titers before performing RIS.

[Immunoscintigraphy in colorectal cancers from the viewpoint of the gastroenterologist]. / Immunszintigraphie bei kolorektalen Karzinomen aus der Sicht des Gastroenterologen.

In the last decade radioimmunoscintigraphy developed from an experimental approach to a clinically useful method. First promising results were obtained with Technetium-99m labelled murine monoclonal antibodies against carcinoembryonic antigen (CEA). Routine clinical application is restricted to colorectal carcinoma at the moment. Radioimmunoscintigraphy is recommended in case of recurring disease when conventional methods are ineffective or doubtful, especially in the pelvic region. The availability of monoclonal antibodies to new tumor-associated antigens will extend the spectrum to other gastrointestinal tumors. The possibility to generate recombinant humanized monoclonal antibodies and antibody fragments are encouraging new perspectives. These developments and the use of biological immune response modifiers to increase antigen expression could provide means to consider radioimmunotherapy of gastrointestinal cancers.