Modification of pK values caused by change in H-bond geometry.

The competition between various groups for a proton is studied by ab initio molecular orbital methods. It is found that reorientations of the two groups involved in a H-bond can reverse the equilibrium position of the proton shared between them. Specifically, the carbonyl and hydroxyl groups were modeled by H2CO and HOH. In the H-bond between these two groups, association of the proton with the carbonyl (H2COH...OH2)+ is favored over the hydroxyl (H2CO...HOH2)+ when the latter group is situated along a lone pair of the carbonyl oxygen. However, displacement of the water to the C = O axis between the two carbonyl lone pairs reverses the situation and (H2CO...HOH2)+ is more stable. A similar reversal of stability is observed in the H-bond involving a Schiff base (modeled by CH2NH) and amine (NH3). In one arrangement where the lone pairs of the two groups point toward one another, the proton prefers the Schiff base to the amine--i.e., (H2CHNH...NH3)+ is more stable than (H2CHN...HNH3)+. On the other hand, rotation of the lone pair of the amine away from the Schiff base nitrogen results in proton transfer across to the amine. These shifts in stability correspond to reversal of relative pK of the groups involved. A fundamental principle emerging from the calculations is that ion-dipole electrostatic interactions favor transfer of a proton to the group that is positioned as closely as possible to the negative end of the dipole moment vector of the other. The ideas developed here suggest a number of means by which conformational changes may be utilized to shift protons from residue to residue within a protein molecule such as an enzyme or bacteriorhodopsin.     

Effects of altered expression and activity levels of CK1δ and ɛ on tumor growth and survival of colorectal cancer patients

Colorectal cancer (CRC) is the fourth leading cause of cancer related death worldwide due to high apoptotic resistance and metastatic potential. Because mutations as well as deregulation of CK1 isoforms contribute to tumor development and tumor progression, CK1 has become an interesting drug target. In this study we show that CK1 isoforms are differently expressed in colon tumor cell lines and that growth of these cell lines can be inhibited by CK1‐specific inhibitors. Furthermore, expression of CK1δ and ? is changed in colorectal tumors compared to normal bowel epithelium, and high CK1? expression levels significantly correlate with prolonged patients' survival. In addition to changes in CK1δ and ? expression, mutations within exon 3 of CK1δ were detected in colorectal tumors. These mutations influence ATP binding resulting in changes in kinetic parameters of CK1δ. Overexpression of these mutants in HT29 cells alters their ability to grow anchorage independently. Consistent with these results, these CK1δ mutants lead to differences in proliferation rate and tumor size in xenografts due to changes in gene expression, especially in genes involved in regulation of cell proliferation, cell cycle, and apoptosis. In summary, our results provide evidence that changes in the expression levels of CK1 isoforms in colorectal tumors correlate with patients' survival. Furthermore, CK1 mutants affect growth and proliferation of tumor cells and induce tumor growth in xenografts, leading to the assumption that CK1 isoforms provide interesting targets for the development of novel effective therapeutic concepts to treat colorectal cancer.     

Evaluation of Myocardial Viability by MRI

Distinguishing between viable and non-viable myocardium is an important clinical issue. Several magnetic resonance (MR) techniques to address this issue have been proposed. Spectroscopy of phosphorus-31 and hydrogen-1 from creatine as well as imaging of sodium-23 and potassium-39 reflect information related to cellular metabolism. The spatial and temporal resolutions of these techniques are limited, however, by the small magnitude of the MR signal. Proton imaging techniques include examination of pathologic alterations in MR relaxation times (T₁ and T₂), wall thickness and thickening, cine MRI combined with low-dose dobutamine, first-pass contrast enhancement patterns, and delayed contrast enhancement patterns. Of the proton imaging approaches, cine MRI combined with low-dose dobutamine is supported by the largest body of clinical evidence supporting the hypothesis that the technique yields useful information regarding myocardial viability. Recent data suggest that delayed contrast enhancement examines the transmural extent of viable myocardium irrespective of contractile function and that this technique should also be considered in a clinical setting. Zusammenfassung Myokardvitalität ist definiert als das Vorliegen lebender Myozyten. Bei Patienten mit ischämischer Herzkrankheit läßt sich die kontraktile Dysfunktion des Herzmuskels sowohl in vitalen als auch in avitalen Gebieten beobachten. In Gebieten mit vitalem Myokard kann sie durch Revaskularisation mit Thrombolytika, perkutaner transluminaler Koronarangioplastie (PTCA) oder koronarer Bypassoperation (CABG) rückgängig gemacht werden. In nekrotischen Arealen ist die kontraktile Dysfunktion dagegen irreversibel, und Revaskularisationsmaßnahmen können aufgrund ihres kleinen, aber eindeutigen Risikos kontraindiziert sein. Die Unterscheidung zwischen vitalen und avitalen Herzmuskelarealen ist daher eine klinisch wichtige Frage. Eine direkte Untersuchung von Patienten auf das Vorliegen bzw. Fehlen lebender Myozyten ist nicht möglich. Daher wird die Myokardvitalität indirekt mit klinischen Verfahren beurteilt. Beispielsweise erfaßt die Echokardiographie mit Dobutaminen Kontraktilitätsänderungen nach inotroper Stimulation, und die ^99mTc-Sestamibi bestimmt die Myokardperfusion. Kernspintomographische Methoden zur Vitalitätsprüfung umfassen spektroskopische und bildgebende Verfahren. Die Phosphor-31- und Wasserstoff-1-MR-Spektroskopie aus Kreatin sowie die Natrium-23- und Kalium-29-Bildgebung liefern unmittelbar mit dem Zellstoffwechsel verbundene Informationen. Die räumliche und die zeitliche Auflösung dieser Verfahren sind jedoch aufgrund des schwachen MR-Signals begrenzt. Bei Wasserprotonen ist das MR-Signal dagegen verhältnismäßig stark und ermöglicht eine Darstellung des Herzens mit hoher räumlicher und zeitlicher Auflösung. Die Bildgebung mit Protonenresonanz beinhaltet die Untersuchung von Änderungen der intrinsischen MR-Relaxationszeiten (T₁ und T₂), Wanddicke und Verschwielung, Cine-MRT mit niedrigdosiertem Dobutamin sowie First-pass- und verzögerte Kontrastverstärkungsmuster. Berichten zufolge kommt es nach ischämischer Schädigung sowohl in vitalen wie in nekrotischen Myokardgebieten zu Änderungen der intrinsischen Relaxationszeiten, was den Nutzen dieses Verfahrens einschränkt. In ähnlicher Weise stehen Änderungen der Wanddicke und/oder Verschwielung nur in begrenztem Zusammenhang mit der Myokardvitalität. Die Cine-MRT mit niedrig dosiertem Dobutamin scheint das intakte Myokard zumindest ebenso gut zu erfassen wie die klinisch bereits etablierte Dobutamin-Echokardiographie. Die regionale Durchblutung, wie sie mittels First-pass-MRT erfasst wird, mag vom Vorhandensein vitalen Myokards abhängen. Die zugrunde liegende Physiologie legt aber nahe, dass Perfusion und Vitalität oft nicht in direkter Beziehung stehen. Neuere Forschungsergebnisse zur verzögerten Kontrastverstärkung weisen auf eine mögliche Definition vitalen Myokards als "Gebiete ohne starke Kontrastmittelaufnahme" hin. Derzeit liegen nur begrenzte klinische Daten vor, die diesen Ansatz unterstützen. Zusammenfassend liegen die aussagekräftigsten klinischen Daten derzeit für die Cine-MRT mit niedrig dosiertem Dobutamin zur Diagnose vitalen Myokards vor. Neueste Ergebnisse lassen vermuten, dass die verzögerte Aufnahme von Kontrastmitteln die transmurale Ausdehnung vitalen Myokards unabhängig von der kontraktilen Funktion gut abbildet und dass diese Technik auch in der Klinik zum Einsatz kommen sollte.     

Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1B binding sites

In rat brain cortex slices preincubated with [3H]5-HT, the potencies of 17 5-HT receptor agonists to inhibit the electrically evoked 3H overflow and the affinities of 13 antagonists (including several beta-adrenoceptor blocking agents) to antagonize competitively the inhibitory effect of unlabelled 5-HT on evoked 3H overflow were determined. The affinities of the compounds for 5-HT1B and 5-HT2 binding sites in rat brain cortex membranes (labelled by [125I]cyanopindolol = [125I]-CYP in the presence of 30 mumol/l isoprenaline and [3H]ketanserin, respectively), for 5-HT1A binding sites in pig and rat brain cortex membranes (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin = [3H]8-OH-DPAT) and for 5-HT1C binding sites in pig choroid plexus membranes (labelled by [3H]mesulergine) were also determined. The affinities of the drugs for the various 5-HT recognition sites ranged over 4-5 log units (the functional experiments revealed the same range of differences between the drugs). There were no significant correlations between the affinities of the drugs at 5-HT1C and 5-HT2 binding sites and their potencies or affinities, determined for the 5-HT autoreceptors. In contrast, significant correlations were found between the potencies or affinities of the drugs for the autoreceptors and their affinities at 5-HT1A or 5-HT1B binding sites; the best correlations were obtained with the 5-HT1B binding site. Some of the drugs investigated were not included in the correlation since their agonistic or antagonistic effects on the autoreceptors were weak and pEC30 or apparent pA2 values could not be determined (less than 5.5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anorectal amelanotic melanoma

Objective  Anorectal melanoma is a rare, highly malignant tumour with a poor 5 year survival of 10%. Most anorectal melanomas have gross and/or histologic pigmentation, however about 30% of anorectal melanomas are amelanotic.
Method  We report three cases of amelanotic anorectal melanomas and integrate our data with six case reports of amelanotic malignant melanoma from the literature. Further we compare clinicopathological data and clinical outcome with large series of anorectal melanomas (both, amelanotic and pigmentated).
Results  There were seven females and two males, of median age 62 years (range: 45–75 years). Rectal bleeding was the leading symptom in all cases with a mean duration of 4 months before diagnosis. Eight of nine patients developed distant metastases. Median survival was 14 months (range: 3–60 months). A tumour thickness of < 4 mm was correlated with long-term disease-free survival, whereas tumour thickness of 4 mm or more was correlated with systemic recurrence.
Conclusion  Early diagnosis is key for efficient treatment and improved survival rate for patients with this unusual variant of melanoma. There is no difference in terms of age, time of diagnosis, stage and survival between pigmented and amelanotic anorectal melanoma.