Comprehensive cancer predisposition testing within the prospective MASTER trial identifies hereditary cancer patients and supports treatment decisions for rare cancers.

BACKGROUND: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers. PATIENTS AND METHODS: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies. RESULTS: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation. CONCLUSIONS: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research.

[New antibiotics for severe infections due to multidrug-resistant pathogens : Definitive treatment and escalation]. / Neue Antibiotika bei schweren Infektionen durch multiresistente Erreger : Definitive Therapie und Eskalation.

Multidrug-resistant pathogens often lead to treatment failure of antimicrobial regimens. After a period of imbalance between the occurrence/spread of resistance mechanisms and the development of new substances, some new substances have meanwhile been approved and many more are currently undergoing clinical testing. They are particularly effective against specific resistance mechanisms/pathogens and should be preserved for definitive treatment of an isolated pathogen. In the absence of alternatives reserve antibiotics, such as aztreonam and colistin have experienced a renaissance. They are again used in special infection scenarios and clinically tested in combination with new substances. Despite the introduction and development of new substances the building of resistance will at some time also render these (at least partially) ineffective. Therefore, their implementation must be carried out according to the antibiotic or infectious diseases stewardship.

Effect of nanoconfinement on polymer dynamics: surface layers and interphases.

We present neutron spin echo experiments that address the much debated topic of dynamic phenomena in polymer melts that are induced by interacting with a confining surface. We find an anchored surface layer that internally is highly mobile and not glassy as heavily promoted in the literature. The polymer dynamics in confinement is, rather, determined by two phases, one fully equal to the bulk polymer and another that is partly anchored at the surface. By strong topological interaction, this phase confines further chains with no direct contact to the surface. These form the often invoked interphase, where the full chain relaxation is impeded through the interaction with the anchored chains.

Effects of phytoestrogen extracts isolated from flax on hormone production of trophoblast tumour cells Jeg 3 and BeWo.

UNLABELLED: AIM AND SETTING: To test the effects of crude extracts from flax (Linum usitatissimum) on progesterone and estradiol and ERα and ß/PR production in choriocarcinoma cell lines Jeg 3 and BeWo. Tumor trophoblast cells (Jeg 3 and BeWo) were incubated in the presence of different concentrations of the flax crude extracts. Estradiol and progesterone production was measured. Estrogen receptor α and ß as well as progesterone receptor expressions were also assessed. RESULTS: In Jeg 3 cells, progesterone production was downregulated by flax root and leaves extract, while in BeWo cells only flax root extract did manage to downregulate progesterone production. ERß expression was significantly downregulated by flax root and flax leaves extract in both cell lines; on the contrary, ERα expression was increased by flax leaves extract in BeWo cells. PR expression was downregulated by flax leaves extract in Jeg 3 and by flax root extract in BeWo cells. CONCLUSION: Flax extracts derived from leaves and especially from roots can modify progesterone and possibly estradiol production, while at the same time they seem to alter ERß expression. Further studies on animal models and adequately designed retrospective epidemiological studies are imperative to clarify this role upon progesterone.

Phytosterols supplementation decreases plasma small and dense LDL levels in metabolic syndrome patients on a westernized type diet.

BACKGROUND AND AIMS: Several studies have observed a hypocholesterolemic effect of plant sterols in hypercholesterolemic patients on a balanced diet. The aim of this study was to examine the effect of phytosterol supplementation on risk factors of coronary artery disease in metabolic syndrome patients on a Westernized type diet. METHODS AND RESULTS: In a randomized placebo-controlled design 108 patients with metabolic syndrome were assigned to consume either 2 plant sterol-enriched yogurt mini drink which provided 4 g phytosterols per day, or a yogurt beverage without phytosterols (control). The duration of the study was 2 months and the patients in both groups followed their habitual westernized type diet and recording it on food diaries. Blood samples were drawn at baseline and after 2 months of intervention. After 2 months supplementation with phytosterols, a significant reduction in total cholesterol, LDL-cholesterol, small and dense LDL (sdLDL) levels, as well as, apoB and triglycerides concentrations were observed in the intervention group (P < 0.05) compared to the control group. In addition, phytosterol supplementation lowered serum total cholesterol by 15.9%, LDL-cholesterol by 20.3% and triglyceride levels by 19.1% (P = 0.02, P < 0.001 and P < 0.001, respectively), although the patients kept their habitual westernized type diet. No differences were observed in HDL cholesterol, apoA1, glucose, C-reactive protein, fibrinogen levels and blood pressure. CONCLUSIONS: Phytosterol supplementation improves risk factors of coronary artery disease even if the diet is a westernized type.

Online monitoring of cellular metabolism in the MCF-7 carcinoma cell line treated with phytoestrogen extracts.

BACKGROUND: Phytoestrogens are naturally occurring, plant-derived, nonsteroidal phytochemicals with anticarcinogenic potential. The aim of this study was to isolate phytoestrogens from the flax root of Linum usitatissimum and to test their effect on cellular metabolism in the human mammalian carcinoma cell line MCF-7 using the Bionas 2500 analysis system. MATERIALS AND METHODS: Metabolically relevant parameters such as acidification, oxygen consumption and cell adhesion were registered continuously over 8 and 24 hours on six sensor chips in parallel at different concentrations of flax root extracts. RESULTS: The extracts from flax roots of L. usitatissimum reduced extracellular acidification, respiration and adhesion in a concentration-dependent manner. CONCLUSION: The Bionas 2500 analysis system allows multiparametric online monitoring of cellular processes and can be used to detect the mode of action of anticarcinogenic compounds in cellular metabolism.

Effects of phytoestrogen extracts isolated from flax on estradiol production and ER/PR expression in MCF7 breast cancer cells.

BACKGROUND: In this study, we tested the effects of crude extracts from flax (Linum usitatissimum) on the production of estradiol and expression of estrogen receptor (ER) and progesterone receptor (PR) in human breast cancer MCF7 cells. MATERIALS AND METHODS: Isoflavone and lignan extracts from flax plant Linum usitatissimum were obtained, using different extraction methods. Breast carcinoma cells (MCF7) were incubated with various concentrations of the isolated extracts. Untreated MCF7 cells were used as controls. Supernatants were removed at designated times and tested for estradiol with an ELISA method. Furthermore, the effect of phytoestrogen extracts on the production of ERa and ERbeta as well as on PR was examined. RESULTS AND CONCLUSION: Production of estradiol is elevated in MCF7 cells in a concentration-dependent manner after stimulation with isoflavone and lignan extracts from Linum usitatissimum. Expression of ERalpha is up-regulated after stimulation with lower concentrations of lignan extracts from flax plants, unchanged at median concentrations and down-regulated at high concentrations. Expression of ERbeta is down-regulated in a concentration-dependent manner.

Environmental gamma dosimetry with OSL of alpha-Al(2)O(3):C for in situ sediment measurements.

The physical properties of alpha-Al(2)O(3):C are very similar to that of quartz, which make it an attractive dosimetric material for geological and archaeological dating applications. Storage experiments in an ultra-low-radiation underground environment (UDO at PTB) and gamma-ray spectrometry show that the optically stimulated luminescence (OSL) signal of this material does neither suffer from a significant inherent background caused by traces of radionuclides (<6 microGy a(-1)) nor from fading. After having performed a simple calibration procedure, gamma dosimetry based on alpha-Al(2)O(3):C detectors, which were exposed in a brick block and a lead castle for different periods of time, provided concordant results with dose values derived from independent gamma-ray spectrometric measurements using high-purity germanium and NaI:Tl detectors. These investigations indirectly confirm both the absence of a significant inherent background and fading of the detector material. Small doses of a few micro gray accumulated in short exposure times to environmental radiation can be accurately measured, even when doses (i.e. transport dose) much larger than the actual environmental dose have to be subtracted. It is shown that the OSL signal caused by small transport doses can be easily and reproducibly reset even under difficult field conditions by illuminating the dosemeters with the blue light from Luxeon LEDs. Summarised, alpha-Al(2)O(3):C appears to be the material of choice for dosimetric dating applications of quartz or related materials, when analysed by using OSL.

[Impact of therapeutic riding on gait and posture regulation]. / Der Einfluss des Therapeutischen Reitens auf den Gang und die Haltungsregulation.

BACKGROUND: Hippotherapy has become an important therapeutic option in patients suffering from motoric dysfunction. The physiologic basis of this approach is the three-dimensional transmission of the horse's motion onto the patients body. These motion stimuli are believed to exert possitve effects on the patients's postural control systems. AIM: To test the hypothesis that hippotherapy has both positive short- and lang-term effects on gait and posture control of persons suffering from motoric disabilities. METHODS: Twenty-two children and adolescents aged 9.69 +/- 4.01 years (range: 9.69 +/- 4.01 years) with motoric dysfunctions were included in a prospective matched control study. In each participant, gait and posture control were investigated on four different occasions (O1 - O4) using the Interactive balance system (IBS; Tetrax Inc., Ramat Gan, Israel) and the portable gait analysis sytem RehaWatch (Hasomed, Magdeburg, Germany). The dates of gait and posture analysis were defined as follows: O 1: immediately prior to first therapeutic riding session (TRS); O 2: immediately after first TRS; O 3: after the last day of an eight weeks period of daily TRS; O 4: seven weeks later after a TRS free interval. RESULTS: The following parameters were slightly improved (adjusted significance level of p < 0.003) after eight weeks of therapeutic riding: (O1 vs. O 3): (1) walking distance (p = 0.009, eta(2) = 0.339); (2) pace frequency (p = 0.007, eta(2) = 0.358); (3) walking speed (p = 0.006, eta(2) = 0.367), and (4) time of attachment (p = 0.007, eta(2) = 0.360). The only short-term effect observed was a significant decrease of the attachment phase (p = 0.002, eta(2) = 0.387). Interestingly, gait symmetry remained unaffected. Posturography (adjusted significance level of p < 0.01) at O 1 versus O 2 (short-term) showed a significant decrease of the performance of both the visual-nigrostriatal subsystem (p < 0.001) and the somato-sensory subsystem (p = 0.001). At O 1 versus O 3 (long-term), the following parameters were sharply decreased: (1) postural stability (p = 0.011), and (2) somatosensory performance (p = 0.011). CONCLUSIONS: In the individuals investigated, an eight weeks series of therapeutic riding did not improve posture control and had only a small positive effect on gait performance. The reasons for these rather disappointing results could have been the low number of therapeutic riding sessions (0.5 sessions per week), and the relatively short duration (30 min) of each session. It remains to be seen, whether a higher density and longer duration of therapeutic riding sessions yields better results.

Effects of phytoestrogen extracts from Linum usitatissimum on the Jeg3 human trophoblast tumour cell line.

BACKGROUND: Phytoestrogens are a diverse group of nonsteroidal plant compounds which have similar effects to endogenous estrogens in humans and have been ascribed potential anticarcinogenic activities. We tested the effects of phytoestrogen extracts from different plant organs of flax, Linum usitatissimum, on cell proliferation in trophoblast tumour cells of the cell line Jeg3. MATERIALS AND METHODS: Phytoestrogen extracts were prepared from leaves, stems and roots of L. usitatissimum using different extraction methods. The isolated phytoestrogens were identified using HPLC-MS analysis. The influence on cell proliferation (MTT test) was determined in the trophoblast tumour cells, Jeg3. RESULTS: Cell proliferation of trophoblast tumour Jeg3 cells was significantly affected by the phytoestrogens isolated from leaves, stems and roots of L. usitatissimum. Root extracts inhibited Jeg3 cell growth significantly. CONCLUSION: A cell culture model system of the human trophoblast tumour cell line, Jeg3, was established to test the effect of potential phytoestrogens on cell proliferation. It was shown that the roots of L. usitatissimum contain measurable concentrations of lignans and isoflavones.

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Flax-seed extracts with phytoestrogenic effects on a hormone receptor-positive tumour cell line.

UNLABELLED: The higher soy intake in the Asian population compared to Europeans is believed to be an essential factor for the lower incidence of hormone-dependent tumours in Asia. It has already been shown that soya beans, with their ingredients genistein and daidzein from the isoflavonoid group, have protective effects on hormone-caused diseases. Lignans are another, less investigated, group of phytoestrogens. The aim of this study was to investigate the effects of flax-seed, which is typically found in Northern European diets, on the proliferation and hormone production of an estrogen receptor (ER)-positive trophoblast tumour cell line. MATERIALS AND METHODS: Trophoblast tumour cells of the cell line Jeg3 were incubated with 2 different concentrations of the isolated crude extract of flax-seed and 7 chemically partitioned extract fractions. Untreated cells were used as controls. After 48 h of stimulation, cell proliferation was measured using the BrdU method. The concentrations of hCG and progesterone produced by the trophoblast tumour cells were measured 48 h after stimulation. Extract fractions with antiproliferative effects in the BrdU- test were analysed by HPLC-MS. RESULTS: Our study showed an inhibitory influence of some of the isolated flax-seed fractions on the Jeg3 tumour cells. Proliferation of the Jeg3 cells was decreased by flax-seed fractions I, V, VI and VII in a dose-dependent manner. Inhibition of hCG production by flax-seed extracts III, V, VI and VII was also dose-dependent. Extract fractions V and VI decreased the production of progesterone by 58% to 86%. Some extract fractions showed a stimulating effect on hormone production and cell proliferation. HPLC-MS analysis showed the presence of matairesinol and biochanin A in flax-seed fraction VI. DISCUSSION: Flax-seed seems to have similar inhibitory effects to soya on hormone production and proliferation of hormone-sensitive tumour cells. Our results showed a dose-dependent inhibition by isolated flax-seed extracts on the Jeg3 cell line. Matairesinol and biochanin A seem to be useful candidates for extended tests on other tumour cell lines and normal tissues to evaluate the potential benefit of a lignan-containing therapy in hormone-dependent diseases.

Effects of phytoestrogens on the trophoblast tumour cell lines BeWo and Jeg3.

INTRODUCTION: Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind to estrogen receptors in humans. With this study we tested the effects of the phytoestrogens genistein and daidzein in cell proliferation and the production of progesterone and hCG in trophoblast tumour cells of the cell lines BeWo and Jeg3. MATERIALS AND METHODS: The phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast tumour cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for progesterone and hCG. In addition we tested the effects of phytoestrogens on cell proliferation. Different concentrations of genistein and daidzein were cultivated with trophoblast tumour cells. After designated times, 1 microCi thymidin-(methyl-3H) was added. Methyl-3H thymidin incorporation was tested and compared to incorporation results of untreated cells. RESULTS: With this study we could show that the production of the steroid hormone progesterone and the protein hormone hCG is influenced by the phytoestrogens genistein and daidzein in trophoblast tumour cells of the cell lines BeWo and Jeg3. We found a correlation between the effects on the proliferation and the production of progesterone and hCG at high concentrations of genistein and daidzein in the cell lines tested. With low concentrations of genistein and daidzein we observed a stimulation of the production of hCG and a weak inhibition of proliferation in both cell lines BeWo and Jeg3. DISCUSSION: The results obtained with this study suggest that only high doses of phytoestrogens (> 1 mumol/ml) can reduce the proliferation of trophoblast tumour cells significantly. Low doses of phytoestrogens induced a higher hCG production in both cell lines tested. Although high hCG production did not lead to a higher proliferation rate of the tumour cells tested, hCG is able to induce neovascularisation in tumour cells. In summary, with this in vitro study we showed that high doses of phytoestrogens inhibit proliferation and progesterone production in trophoblast tumour cells. High doses of phytoestrogens could be useful candidates for special diet programs for prevention and surgery for patients with this type of disease. In addition we found a useful cell culture model for the testing of new types of phytoestrogens.

Immunohistochemical expression of steroid receptors and glycodelin A in isolated proliferative human endometrial glandular cells after stimulation with tamoxifen and phytoestrogens (genistein and daidzein).

INTRODUCTION: The aims of this study were an evaluation of the distribution patterns of steroid hormone receptors (ER, PR) and glycodelin A (GdA) expression of proliferative endometrial glandular cells after stimulation with tamoxifen (TAM) and phytoestrogens (PE) (genistein, daidzein). MATERIALS AND METHODS: Human endometrium was obtained from 4 premenopausal women. Glands were stimulated after isolation with single doses of TAM, genistein and daizein (0.1, 1 and 10 mumol/l) and characterised with ER, PR and GdA after 9 days of culture. RESULTS: ER showed a significant decline with the highest TAM and genistein concentration (p < 0.05), whereas PR increased significantly with TAM and genistein concentrations of 1 mumol/l and 10 mumol/l (p < 0.05). GdA did not show any significant expression under TAM and genistein stimulation. Stimulation with daidzein resulted in no statistically relevant alterations in ER, whereas the PR significantly increased with all three concentrations (p < 0.05) and GdA also showed a significant increase with 1 mumol/l (p < 0.05). DISCUSSION: TAM showed anti-estrogenic properties in premenopausal endometrium. PE showed a similar ER, PR expression pattern as TAM, so therefore PE (genistein and daidzein) could also act as antiestrogens. GdA marked a cell transformation from proliferative to secretory status or the antiestrogen effects of TAM and PE.

Identification of a cis-acting dendritic targeting element in the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II.

In mammalian neurons a selected group of mRNAs, including the transcript encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II, is found in dendrites. The molecular mechanisms underlying extrasomatic RNA trafficking are not well described. It is thought that dendritic transcripts contain cis-acting elements that direct their selective subcellular sorting. Here we report the identification of an extrasomatic targeting element in the 3' untranslated region of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. In primary hippocampal neurons, this 1200-nucleotide-spanning, cis-acting element is sufficient to mediate dendritic localization of chimeric reporter transcripts. The trafficking signal does not share any striking sequence similarity with a previously characterized dendritic targeting element in transcripts encoding the microtubule-associated protein 2. In dendrites of transfected primary neurons, recombinant RNAs form granules with an average diameter of 0.45 microm that may represent preferential RNA docking sites or multimolecular transport units. These findings imply that extrasomatic sorting of individual dendritic mRNAs involves at least partially distinct molecular mechanisms, as well as large trafficking complexes.

VP-RBP, a protein enriched in brain tissue, specifically interacts with the dendritic localizer sequence of rat vasopressin mRNA.

The concept of mRNA localization suggests that this process is mediated by sequences residing in the transcript to which proteins specifically bind and ultimately deliver the mRNA along cytoskeletal elements to specific intracellular destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes both in hypothalamic magnocellular neurons and in primary cultured neurons derived from embryonic rat superior cervical ganglia microinjected with a corresponding eukaryotic expression vector. The last 395 nucleotides of the VP mRNA encompassing part of the coding region, as well as the complete 3'-untranslated region, are sufficient to confer dendritic targeting to a normally nonlocalized reporter transcript. Here we report that, by employing in vitro crosslinking analyses with rat brain proteins and radiolabelled VP transcripts, an RNA-binding protein specifically interacts with the dendritic localizer sequence of the VP mRNA. This protein is enriched in nerve cell tissues. Peripheral tissues and various cell lines contain only low amounts of the binding activity. It therefore represents a candidate protein that may be involved in any aspect related to subcellular VP mRNA sorting in nerve cells, including transport and anchoring of the mRNA and possibly its translational control.

Perceptions of alternative therapies available for women facing hysterectomy or menopause.

Women's perceptions of alternative therapies available for managing symptoms related to menopause and to the need for hysterectomy were explored. Data are reported from 17 focus groups of peri- and menopausal aged women (n = 82) living in two southern U.S. coastal counties. Analysis showed that emergent themes did not vary by race or surgery experience. Herbal preparations, vitamin supplements, "healthy living" (diet & exercise), and mind/body practices (prayer & "mental healing") were mentioned as possible alternatives for managing symptoms. Participants more frequently identified "other" conventional medical approaches--prescription medication, laser surgery, dilation and curettage, and "watchful waiting"--as alternatives to hysterectomy. Alternative therapies are linked to women's desire to gain control over their own health care decisions and practices.

Anoxic ATP depletion in neonatal mice brainstem is prevented by creatine supplementation.

BACKGROUND: Sufficient ATP concentrations maintain physiological processes and protect tissue from hypoxic damage. With decreasing oxygen concentration, ATP synthesis relies increasingly on the presence of phosphocreatine. AIM: The effect of exogenously applied creatine on phosphocreatine and ATP concentrations was studied under control and anoxic conditions. METHODS: Pregnant mice were fed orally with creatine monohydrate (2 g/kg body weight/day). Brainstem slices from these mice pups were compared with those from pups of non-creatine supplemented pregnant mice. Measurements were performed under normoxic and anoxic conditions. In addition, brainstem slices from non-creatine treated mice pups were incubated for 3 hours in control artificial cerebrospinal fluid (CSF) (n = 10) or in artificial CSF containing 200 microM creatine (n = 10). ATP and phosphocreatine contents were determined enzymatically in single brainstem slices. RESULTS: ATP concentrations were in the same range in all preparations. However, there was a significant increase of phosphocreatine in the brainstems from pups of creatine fed mice when compared with the brainstems of pups from non-creatine treated mice or in non-incubated brainstems of control animals. After 30 minutes anoxia, ATP as well as phosphocreatine concentrations remained significantly higher in creatine pretreated slices compared with controls. CONCLUSION: The data indicate that exogenous application of creatine is effective in neuroprotection.

The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family.

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Rat somatostatin receptor subtype 4 can be made sensitive to agonist-induced internalization by mutation of a single threonine (residue 331).

A sequence motif of 20 amino acid residues within the C-terminal portion of the rat somatostatin receptor subtype 4 (SSTR4) has been shown to prevent rapid agonist-dependent receptor internalization in transfected human embryonic kidney (HEK) cells. Molecular dissection of this motif by biochemical ligand-binding assays revealed that the block was released by mutating a single residue (threonine 331) to an alanine. These data are in line with confocal microscopic analysis of cultured primary neurons microinjected with cDNA constructs encoding either SSTR4 or the mutant T331A. Immunocytochemical analysis showed that the mutant receptor, but not SSTR4, was internalized. However, internalized T331A was not recycled to the cell surface, suggesting that it lacks sequence elements that determine intracellular sorting after endocytosis. Neither wildtype SSTR nor the mutant T331A exhibited functional desensitization when assayed for their ability to inhibit adenylate cyclase. In agreement with this, the wt receptor and its mutant were not phosphorylated in response to agonist treatment. Lack of desensitization of SSTR4 has been electrophysiologically verified by coexpressing the receptor with a G-protein-gated, inwardly rectifying potassium channel in Xenopus oocytes. A strong somatostatin 14 (SST14)-activated inward potassium current was observed that was long-lasting and which decayed only slowly after washout of the agonist. This is in contrast to another somatostatin receptor subtype, SSTR3, which mediates rapidly desensitizing currents. Binding experiments on HEK cells transfected with either SSTR3 or 4 indicated that this difference is not attributable to slow dissociation of the agonist from the receptor, suggesting that SSTR4 mediates long-lasting signalling, a property which may be relevant for clinical therapy.