Development of novel 68Ga- and 18F-labeled GnRH-I analogues with high GnRHR-targeting efficiency

A large majority of tumors of the reproductive system express the gonadotropin releasing hormone receptor (GnRHR). Blockade and activation of this receptor with various antagonistic and agonistic analogues of native GnRH-I (pGlu(1)-His(2)-Trp(3)-Ser(4)-Tyr(5)-Gly (6)-Leu(7)-Arg(8)-Pro(9)-Gly(10)-NH2), respectively, has shown efficient suppression of tumor growth. In this study, the GnRH-receptor system has been evaluated with respect to its suitability as a target for in vivo peptide receptor targeting using radiolabeled GnRH-analogues, and in parallel, new (18)F- and (68Ga)-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [(125)I]Triptorelin (D-Trp(6)-GnRH-I) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (approximately 3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/internalization. However, substitution of fetal calf serum by NU serum in the culture medium led to increased and stable GnRHR-expression, especially in the ovarian cancer cell line EFO-27, thus allowing for a stable experimental setup for the evaluation of the new radiolabeled GnRH-I analogues. The new radiolabeled GnRH-I analogues developed in this study were all based on the D-Lys(6)-GnRH-I-scaffold. For (68)Ga-labeling, the latter was coupled with DOTA at D-Lys(6). To allow (18)F-labeling via chemoselective oxime formation, D-Lys(6)-GnRH-I was also conjugated with Ahx (aminohexanoic acid) or beta-Ala, which in turn was coupled with Boc-aminooxyacetic acid. (18)F-labeling via oxime formation with 4-[(18)F]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [(68)Ga]DOTA-GnRH-I, D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I, and D-Lys(6)-betaAla([(18)F]FBOA)-GnRH-I (FBOA = fluorobenzyloxime acetyl) were determined using GnRHR-membrane preparations, and internalization efficiency of the new radioligands was determined in EFO-27 cells. Both quantities were highest for D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I (IC 50 = 0.50 +/- 0.08 nM vs 0.13 +/- 0.08 nM for Triptorelin; internalization: 86 +/- 16% of the internal reference [(125)I]Triptorelin), already substantially reduced in the case of the -betaAla([(18)F]FBOA)-derivative (IC 50 = 0.86 +/- 0.13 nM; internalization: 42 +/- 3% of [(125)I]Triptorelin), while the [(68)Ga]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC50 = 13.3 +/- 1.0 nM; internalization: 2.6 +/- 1.0% of [(125)I]Triptorelin). Generally, the lipophilic residue [(18)F]FBOA is much better tolerated as a modification of the D-Lys(6)-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys(6)-side chain and the [(18)F]FBOA-moiety. In summary, D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I shows the highest potential for efficient GnRHR-targeting in vivo of the compounds investigated. Unfortunately, however, the very low cell surface expression of GnRH-receptors and thus very low radioligand uptake by GnRHR-positive tumor cells found in vitro was also confirmed by a preliminary biodistribution study in OVCAR-3 xenografted nude mice using the standard GnRHR radioligand [(125)I]Triptorelin. Tumor uptake was lower than blood activity concentration at 1 h p.i. (0.49 +/- 0.05 vs 0.96 +/- 0.13 for tumor and blood, respectively). These data seriously challenge the suitability of the GnRHR-system as a suitable target for in vivo peptide receptor imaging using radiolabeled GnRH-I derivatives, despite the availability of high-affinity radiolabeled receptor-ligands such as D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I.     

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1‐3 pskr2‐1 and tpst‐1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1‐3 pskr2‐1 and of tpst‐1 were shorter. In planta, growth of wild‐type pollen in pskr1‐3 pskr2‐1 and tpst‐1 flowers appeared slower than growth in wild‐type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1‐3 pskr2‐1 and in tpst‐1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross‐pollination experiments with wild‐type, pskr1‐3 pskr2‐1 and tpst‐1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.     

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein–protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.     

Pleasure and disgust: memories and associations of pleasant and unpleasant odours in Germany and Japan

The question of whether odour experiences have a function inthe cultures of the technically industrialized world was investigatedby interviews with German (166) and Japanese (88) subjects.They were asked to name pleasant and unpleasant odours frommemory and to give associations regarding them. The participantsenumerated 2040 odours and 3520 associations. Although Japanesesubjects in general gave less information than the Germans,the results for both cultures were very similar in quality aswell as quantity. The odour memories included the whole physicaland social environment and the associations showed the far-reachingeffect of odour experiences. Both pleasant and unpleasant odourswere remembered to an equal extent. Most of the odours recalledwere assessed similarly by the subjects, as either pleasantor unpleasant. Obviously, individual likings or aversions aredominated by underlying evaluations common to all subjects ofboth cultures. Uniformly judged odours were, for example, odoursof plants (pleasant) and odours of rotten and decomposed things(unpleasant). It is discussed whether this finding reflectspreprogrammed survival strategies. The few cultural differenceswhich were found seemed to reflect different life habits, normsand values in the two countries.     

Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V₁ sector catalyzes ATP hydrolysis and the V₀ sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V₀ assembly. We have discovered a fifth V₀ assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V₀ assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V₀–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V₀ subunits c, c′, and c″. On assembly of the remaining three V₀ subunits (a, d, and e) into the V₀ complex, Voa1p dissociates from the now fully assembled V₀–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V₀ assembly in the ER, but then it dissociates before exit of the V₀–Vma21p complex from the ER for transport to the Golgi compartment.     

The in vivo effects of neutralizing antibodies against IFN-γ, IL-4, or IL-10 on the humoral immune response in young and aged mice

In the present study we investigated whether age-related changes in the composition and functional properties of murine CD4⁺ T cells are reflected in vivo by a changed humoral response to influenza vaccine in aged mice. After the primary immunization, the titers of influenza-specific IgM, IgG1, IgG2a, and IgG2b, but not of IgG3 and IgE, were significantly reduced in aged mice compared to young mice. Treatment of aged mice with anti-IFN-γ, anti-IL-4, or anti-IL-10 resulted in levels of IgM and IgG1 comparable to those found in young mice, whereas IgG2a and IgG2b were further decreased. After the booster immunization IgE was significantly enhanced in aged mice, whereas no differences were observed with regard to the other isotypes. During the primary response in young mice, anti-IFN-γ stimulated IgG1 and IgE, whereas an inhibition of IgG2a, IgG2b, and IgG3 was observed. Anti-IL-4 caused a decrease only in IgG3 while anti-IL-10 increased IgM and IgG1 and decreased IgG2b and IgG3. During the primary response in aged mice, all anti-cytokine antibodies enhanced IgM and IgG1 while IgE was only enhanced by anti-IL-10. By contrast, IgG3 was inhibited by anti-IFN-γ and anti-IL-10. Anti-cytokine treatment of young mice increased all isotypes, except IgG3, in the secondary response, whereas the secondary response in aged mice was largely insensitive to anti-cytokine treatment. These data therefore support the idea that the in vivo effects of cytokines on isotype switching are dependent on the differentiation stage of B cells which may be different in young and aged mice.     

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.     

Levels of {beta}-Glucan and Lignin in Elongating Internodes of Deepwater Rice

Partial submergence or treatment with either ethylene or gibberellicacid (GA₃ induces rapid growth in deepwater rice (Oryza sativaL.). We correlated the synthesis of two cell wall componentswith two phases of internodal elongation, namely (13,14)-ß-glucanformation with cell elongation and lignification with differentiationof the secondary cell wall and cessation of growth. The contentof ß-glucan was highest in the zone of cell elongationin internodes of air-grown plants and plants that were inducedto grow rapidly by submergence. In the intercalary meristemand in the differentiation zone of the internode, ß-glucanlevels were ca. 70% lower than in the zone of cell elongation.The outer cell layers, enriched in epidermis, contained moreß-glucan in submerged, rapidly growing internodesthan in air-grown, control internodes. The ß-glucancontent of the inner, parenchymal tissue was unaffected or slightlylowered by submergence. The epidermis appears to be the growth-limitingstructure of rapidly growing rice internodes. We hypothesizethat elevated levels of ß-glucan contribute to elongationgrowth by increasing the extensibility of the cell wall. Lignificationwas monitored by measuring the content of lignin and the activitiesof two enzymes of the lignin biosynthetic pathway, coniferylalcohol dehydrogenase (CAD) and phenylalanine ammonia-lyase(PAL), in growing and non-growing regions of the internode.Using submerged whole plants and GA₃-treated excised stem segments,we showed that lignin content and CAD activity were up to sixfoldlower in newly formed internodal tissue of rapidly growing ricethan in slowly growing tissue. No differences were observedin parts of the internode that had been formed prior to inductionof growth. PAL activity was reduced throughout the internodeof submerged plants. We conclude that lignification is one ofthe processes that is suppressed to permit rapid growth. ¹ This work was supported by the National Science Foundationthrough grants No. DCB-8718873 and DCB-9103747 and by the Departmentof Energy through grant No. DE-FGO2-90ER20021. M.S. was therecipient of a fellowship from the Max Kade Foundation.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.