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Accumulating evidence suggests that p53 plays a suppressive role in cancer metastasis, yet the underlying mechanism remains largely unclear. Regulation of actin dynamics is essential for the control of cell migration, which is an important step in metastasis. The Arp2/3 complex is a major nucleation factor to initiate branched actin polymerization that drives cell migration. However, it is unknown whether p53 could suppress metastasis through modulating Arp2/3 function. Here, we report that WDR63 is transcriptionally upregulated by p53. We show with migration assays and mouse xenograft models that WDR63 negatively regulates cell migration, invasion, and metastasis downstream of p53. Mechanistically, WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3‐mediated actin polymerization. Furthermore, WDR63 overexpression is sufficient to dampen the increase in cell migration, invasion, and metastasis induced by p53 depletion. Together, these findings suggest that WDR63 is an important player in the regulation of Arp2/3 function and also implicate WDR63 as a critical mediator of p53 in suppressing metastasis.

In this contribution, we report three perylenediimides (PDIs) substituted with hydrogen (PDI), mono-nitro (PDIN) and di-nitro groups (PDI2N) at bay positions. The resulting sensing devices, which were composed of self-assembled micro/nanoribbons, exhibited high sensitivity toward ammonia vapor. Among the three PDIs, the PDI2N device displayed the lowest detection limit, as evidenced by its reduction potential of −0.36 eV, and the best ambient stability due to its reduction potential being as low as −4.35 eV. A positive reduction potential and moderate π–π orbital overlap conferred the PDIN device with the highest sensitivity. By contrast, a negative reduction potential of −0.73 eV resulted in a severe lack of carrier density and, therefore, induced the poorest sensitivity in the PDIs device. These results reveal that detection limits are closely associated with the reduction potential of PDIs devices and that ambient stability is determined by the lowest unoccupied molecular orbital energy level. The combined effects of reduction potential and π–π orbital overlap modulated the sensitivity of the PDIs devices. This study not only presents sensitive and ambient-stable PDIs sensing devices but also identifies the critical factors influencing their sensitivity, detection limit, and stability. The excellent sensing performance and extensive theoretical research presented in this work are expected to provide a basis for the design of simple, efficient, and highly stable PDIs sensors.

The presence and functions of nuclear actin have been controversial due to the lack of molecular mechanisms. Nuclear actin and actin-related proteins (Arps) are subunits of several chromatin remodelers, including the evolutionarily conserved INO80 chromatin-remodeling complex. Here, we present an improved cryo-EM structure of the yeast INO80 complex and the first 3D reconstruction of the INO80 actin/Arp module. The modular and subunit architecture is defined using a combination of subunit deletion analysis and published crosslinking-mass spectrometry. The functional interactions of the INO80 actin/Arp module with a nucleosome is 3D EM reconstructed in two different binding states. Nucleosomes initially bind to the Arp8 subunit and the substantial conformational changes maximize nucleosome contacts of the actin/Arp module, which could promote the bound nucleosome to be engaged onto the INO80 ATPase domain. Our findings suggest that the conserved nuclear actin/Arp module acts a conformational switch of the INO80 for nucleosome binding.

The NuA4/TIP60 acetyltransferase complex is required for gene regulation, DNA repair and cell cycle progression. The limited structural information impeded understanding of NuA4/TIP60 assembly and regulatory mechanism. Here, we report the 4.7 Å cryo-electron microscopy (cryo-EM) structure of a NuA4/TIP60 TEEAA assembly (Tra1, Eaf1, Eaf5, actin and Arp4) and the 7.6 Å cryo-EM structure of a TEEAA-piccolo assembly (Esa1, Epl1, Yng2 and Eaf6). The Tra1 and Eaf1 constitute the assembly scaffold. The Eaf1 SANT domain tightly binds to the LBE and FATC domains of Tra1 by ionic interactions. The actin/Arp4 peripherally associates with Eaf1 HSA domain. The Eaf5/7/3 (TINTIN) and piccolo modules largely pack against the FAT and HEAT repeats of Tra1 and their association depends on Eaf1 N-terminal and HSA regions, respectively. These structures elucidate the detailed architecture and molecular interactions between NuA4 subunits and offer exciting insights into the scaffolding and regulatory mechanisms of Tra1 pseudokinase., The NuA4 histone acetyltransferase complex is important for gene regulation, DNA repair processes and cell cycle progression. Here the authors give molecular insights into the NuA4 complex by presenting the cryo-EM structures of the NuA4 TEEAA (Tra1, Eaf1, Eaf5, actin, and Arp4) and TEEAA-piccolo NuA4 assemblies.

Maxillary sinus lift surgery (MSLS) is considered to be a useful treatment method for patients with atrophic alveolar bone. Choosing a suitable surgical procedure to simultaneously decrease graft absorption and obtain long-term survival of dental implants is still a challenge. In this study, 20 patients received MSLS with graft of bone morphogenetic protein 2 (BMP2)-loaded calcium phosphate. Dental implants were placed simultaneously in 10 patients receiving MSLS (1-stage group), and in 10 patients receiving dental implants with a 3 to 6 months delay (2-stage group). The effects were evaluated based on clinical and radiographic examination during a 4 to 5 years follow-up. The results showed that only 1 perforation of the maxillary sinus mucosa was observed in 1-stage group, and was patched with a collagen membrane. An average bone gain of 6.8 mm was observed, and all implants exhibited no looseness, peri-implantitis, or fracture, all of which were stable during the follow-up and exhibited nice dental function during a 4 to 5 years follow-up. The loss of peri-implant bone height was 1.12 ± 0.47 and 1.10 ± 0.39 mm, the probing depth of periodontal pocket was 1.79 ± 0.62 and 1.81 ± 0.71 mm, the sulcular bleeding index was 1.63 ± 0.47 and 1.72 ± 0.54 in 1-stage group and 2-stage group, respectively, and no significant differences existed between these 2 groups. These findings implied that BMP2-loaded calcium phosphate may be a suitable material for MSLS, especially for patients with minimal bone height. Clinicians can use the 1- or 2-stage technique based on clinical condition, patients' choice and clinicians' experience. In patients where implants cannot be stabilized for patients with minimal bone height, 2-stage surgery may be more suitable.

The detection of ammonia vapor has strong implications for health, safety and environment. Herein, we reported on an high efficient chemiresistive sensor based on perylenediimide derivatives (PDIs) substituted by (S)-1-octyn-3-ol (SOT), (S)-3-butyn-2-ol (STB) and (S)-1-phenyl-2-propyn-1-ol (SPP) on 1, 7- bay positions. These PDIs devices exhibited high sensitivity, excellent selectivity and low detection limit towards ammonia vapor. In these materials, SPP showed the most efficient sensing performance, and the increment of current is 4 and 1.2 orders of magnitude higher than SOT and STB due to its lowest LUMO energy, uniform and continuous crystalline phase with π-π stacking distance of 3.28 A. While the highest LUMO energy, poor crystallinity, and smaller π-π overlap as well as largest π-π distance of 3.35 A resulted in the lowest performance of SOT device. It is expected that excellent sensing parameters and detailed analysis of the roles of core-substituted chiral groups will provide a basis protocol for the design of simple, low-power and high efficient PDIs sensors.

Leucine aminopeptidase 3 is involved in the progression and metastasis of several cancers. This study aimed to screen anti-tumor lead compounds targeting leucine aminopeptidase 3. The compounds' suppression effect on enzyme activity and anti-tumor activity were evaluated through a series of assays. Leucine aminopeptidase 3 overexpression K562 cells were used as an enzyme source to screen 43 natural marine compounds. Compounds 5 and 6 exhibited high suppression effect on leucine aminopeptidase 3 activity. Cell activity tests indicated that both compounds have an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay and transwell invasion assay showed that both compounds could inhibit the migration and invasion of breast cancer cells. Immunoblot analysis exhibited that both compounds could downregulate the expression of metastasis-related proteins fascin and matrix metalloproteinase-2/9. A molecular dynamic simulation process was applied to discover the key features of compounds 5 and 6 in binding to leucine aminopeptidase 3 active site. This study described the anti-tumor effects of two leucine aminopeptidase 3 small molecule inhibitors. Taken together, compounds 5 and 6 could be used as anti-tumor lead compounds targeting leucine aminopeptidase 3.

The Saccharomyces cerevisiae Mec1 kinase, the mammalian ATR ortholog, is essential for sensing a variety of DNA lesions and initiating DNA damage response. The Dpb11, a homolog of human TopBP1, functions in activating the Mec1 upon DNA replication stress and DNA damages. Here, we report an affinity purification and ion exchange chromatography method to efficiently purify endogenous Dpb11 under normal expression level directly from yeast whole cell extraction. The final concentration of 5 μM of high purity and homogeneity biochemical preparation enables functional and structural characterization of the physical interaction between Dpb11 and Mec1–Ddc2 complex. The Dpb11 obtained by endogenous purification strongly stimulates the Mec1 kinase activity and promotes the changes in conformational distribution. This observation suggests the Dpb11 activates Mec1 kinase probably through modulation in the kinase conformations.

Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.

Structures of eukaryotic ribonuclease P Ribonuclease P (RNase P) is a ribozyme that processes transfer RNA (tRNA) precursors and is found in all three kingdoms of life. Now, Lan et al. report the structures of yeast RNase P (see the Perspective by Scott and Nagai). The aporibozyme structure reveals how the protein components stabilize the RNA and explains how the structural roles of bacterial RNA elements have been delegated to the protein components in RNase P of higher organisms during evolution. The structure of yeast RNase P in complex with its natural substrate, a tRNA precursor, demonstrates the structural basis for substrate recognition and provides insights into its catalytic mechanism. Science , this issue p. eaat6678 ; see also p. 644

Multidrug resistance (MDR) of hepatocellular carcinoma is a serious problem. Although CD13 is a biomarker in human liver cancer stem cells, the relationship between CD13 and MDR remains uncertain. This study uses liver cancer cell model to understand the role of CD13 in enhancing the cytotoxic effect of chemotherapy agents. Cytotoxic agents can induce CD13 expression. CD13 inhibitor, bestatin, enhances the antitumor effect of cytotoxic agents. Meanwhile, CD13-targeting siRNA and neutralizing antibody can enhance the cytotoxic effect of 5-fluorouracil (5FU). CD13 overexpression increases cell survival upon cytotoxic agents treatment, while the knockdown of CD13 causes hypersensitivity of cells to cytotoxic agents treatment. Mechanistically, the inhibition of CD13 leads to the increase of cellular reactive oxygen species (ROS). BC-02 is a novel mutual prodrug (hybrid drug) of bestatin and 5FU. Notably, BC-02 can inhibit cellular activity in both parental and drug-resistant cells, accompanied with significantly increased ROS level. Moreover, the survival time of Kunming mice bearing H22 cells under BC-02 treatment is comparable to the capecitabine treatment at maximum dosage. These data implicate a therapeutic method to reverse MDR by targeting CD13, and indicate that BC-02 is a potent antitumor compound.

Purpose: A number of 111In- and 99mTc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A 68Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of 68Ga from a generator. The aim of the study was to develop a new 68Ga-folate-based PET radiotracer. Methods: Two new DOTA-folate conjugates, named P3026 and P1254, were synthesized using the 1,2-diaminoethane and 3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propylamine as a spacer, respectively. Both conjugates were labelled with 67/68Ga. Binding affinity, internalization and externalization studies were performed using the FR-positive KB cell line. Biodistribution and PET/CT imaging studies were performed in nude mice, on a folate-deficient diet, bearing KB and HT1080 (FR-negative) tumours, concurrently. The new radiotracers were evaluated comparatively to the reference molecule 111In-DTPA-folate (111In-P3139). Results: The Kd values of 67/68Ga-P3026 (4.65 ± 0.82 nM) and 67/68Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order 67/68Ga-P3026 > 67/68Ga-P1254 > 111In-P3139, while almost double cellular retention was found for 67/68Ga-P3026 and 67/68Ga-P1254, compared to 111In-P3139. The biodistribution data of 67/68Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to 111In-P3139. PET/CT images, performed with 68Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours. Conclusion: The DOTA-folate conjugates can be efficiently labelled with 68Ga in labelling yields and specific activities which allow clinical application. The characteristics of the 67/68Ga-DOTA-folates are comparable to 111In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers for FR-positive tumours

Objective To investigate the effect of fluorescent dye labeling on the targeting capabilities of 111In- (DTPA)n-trastuzumab-(IRDye 800)m. Methods Trastuzumab-based conjugates were synthesized and conjugated with diethylenetriaminepentaacetic acid (DTPA) at molar ratios of 1, 2, 3 and 5 and with a fluorescent dye (IRDye 800CW) at molar ratios of 1, 3 and 5. Immunoreactivity and internalization were assessed on SKBR-3 cells, overexpressing human epidermal growth factor receptor 2. The stability in human serum and phosphate-buffered saline (PBS) was evaluated. The biodistribution of dual-labeled conjugates was compared with that of 111In-(DTPA)2-trastuzumab in a SKBR-3 xenograft model to evaluate the effect of dye-to-protein ratio. Results All trastuzumab-based conjugates exhibited a high level of chemical and optical purity. Flow cytometry results showed that increasing dye-to-protein ratios were associated with decreased immunoreactivity. Stability studies revealed that the conjugate was stable in PBS, while in human serum, increased degradation and protein precipitation were observed with increasing dye-to-protein ratios. At 4 h, the percentages of internalization of dual-labeled conjugates normalized by dye-to-protein ratio (m) were 24.88%±2.10%, 19.99%±0.59%, and 17.47%±1.26% for "m" equal to 1, 3, and 5, respectively. A biodistribution study revealed a progressive decrease in tumor uptake with an increase in the dye-to-protein ratios. The liver, spleen and kidney showed a marked uptake with increased dye-to-protein ratios, particularly in the latter. Conclusions With non-specific-site conjugation of the fluorescent dye with a protein based on imaging agent, the increase in dye-to-protein ratios negatively impacted the immunoreactivity and stability, indicating a reduced tumor uptake.

This study aimed at analyzing the cytotoxicity and pathological effects of cadmium on the ovary. Our studies revealed that cadmium was deposited in the mouse ovary after 8 d cadmium injection in vivo. Also, the increase in the rate of body weight was slowed, while the gonadosomatic index was reduced in the CdCl2 group, compared with the control group. Meanwhile, cadmium affected the maturation of follicles, the degradation of corpus luteum, the arrangement of follicles and corpus luteum, and increased the number of atresia follicles. Besides, under the electron microscope, chromatin margination, karopyknosis, swelling of mature cisternae of Golgi apparatus, mitochondrial cristae disappearance, and swelling of the rough endoplasmic reticulum can be observed in the CdCl2 group mice. Collectively, our findings elucidated the morphological mechanism that the exposure of cadmium changed the ultrastructure of cells in ovary tissues.

Stomatal number and stomatal conductance are important structural and functional parameters for the assessment of carbon assimilation and water use under elevated CO2. We studied stomatal density, number of stomatal rows and stomatal conductance of Pinus sylvestriformis and P. koraiensis needles exposed to elevated CO2 (500 μmol mol−1 CO2) in open-top chambers for 10 years (1999–2009). Elevated CO2 increased stomatal density on P. sylvestriformis by 10.8 % (13.5 % on abaxial surface and 8.0 % on adaxial surface) and the number of stomatal rows on P. koraiensis by 7.9 % (5.0 % in 1-year-old needles and 10.7 % in current-year needles). Increased stomatal density for P. sylvestriformis and number of stomatal rows for P. koraiensis indicate that elevated CO2 increases stomatal number in both tree species. Needle age significantly influenced stomatal density and number of stomatal rows in P. koraiensis but not in P. sylvestriformis. For both species, elevated CO2 did not significantly affect stomatal conductance but increased water use efficiency. The increase in stomatal number is not accompanied by significant changes in stomatal conductance at elevated CO2 for both tree species suggesting that there may be no direct relationship between stomatal conductance and stomatal numbers.

Staring from l -lactide and e-caprolactone, the corresponding lactic-caprolactone cooligomer with hydroxyl and carboxylic acid groups were synthesized. These oligomers were connected with chelating groups through a long chain tether, ready for transition metal binding. Coordination of organometallic rhenium(I) and technetium(I) complexes generated the conjugates in high yield and short time, satisfying the requirements for short-lived radiopharmaceuticals in clinical applications. A reasonable pharmacophore model has been established to guide the design of well-defined lactic acid oligomer for nuclear medicine.

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga.RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC(50) and K(d) values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca(2+) mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with (111)In-RM2 and (68)Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with (68)Ga-RM2.RM2 and (111)In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7 ± 3.3 nmol/l for RM2; 9.3 ± 3.3 nmol/l for (nat)In-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca(2+) mobilization assays. (68)Ga- and (111)In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2 ± 4.8%IA/g at 1 h; 11.7 ± 2.4%IA/g at 4 h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6 ± 4.7%IA/g at 1 h to 1.5 ± 0.5%IA/g at 4 h.RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that (111)In-RM2 and (68)Ga-RM2 are ideal candidates for clinical SPECT and PET studies.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

Recent preclinical and clinical studies show that dyes that excite and fluoresce in the near-infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic.Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800 CW in (DTPA)(n)-trastuzumab-(IRDye 800)(m) conjugate sample preparations in which high-performance liquid chromatography (HPLC) assessment of free dye was not possible.The SDS-PAGE assay was accurate and valid for free IRDye 800 CW amounts between 38 and 4 mol% of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined.This method may be applicable to other near-infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging.

Purpose: G protein–coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR).Experimental Design: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy.Results: The IC50 value of [natIn]-RM1 was 14 ± 3.4 nmol/L. [nat/111In]-RM1 was found to bind to the GRPR with a Kd of 8.5 ± 2.7 nmol/L compared with a Kd of 0.6 ± 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 ± 0.2 nmol/L) compared with [111In]-AMBA (0.7 ± 0.1 nmol/L). [natLu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [natIn]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 ± 0.8% IA/g versus 3.69 ± 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios.Conclusion: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor–targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors. (Clin Cancer Res 2009;15(16):5240–9)

PURPOSE: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available beta-emitting radiometals due to low complex stability. Low tumour-to-kidney ratio of the known radiopharmaceuticals is further limiting their potency. We used macrocyclic chelators for coupling to increase complex stability, modified the peptide sequence to enhance radiolytic stability and studied tumour-to-kidney ratio and metabolic stability using (111)In-labelled derivatives. METHODS: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using (111)In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the (nat)In-metallated compounds were determined by receptor autoradiography using (125)I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the (111)In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. RESULTS: IC(50) values of the (nat)In-metallated gastrin derivatives vary between 1.2 and 4.8 nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC(50) between 9.9 and 1,195 nmol/L). All cholecystokinin receptor affinities were >100 nmol/L. All (111)In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6 h, positively correlating with the number of Glu residues. All (111)In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24 h (0.37-0.99) compared to (111)In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24 h was low. Imaging studies confirmed receptor-specific blocking of the tumour uptake. CONCLUSIONS: Reducing the number of glutamates increased tumour-to-kidney ratio but resulted in lower metabolic stability. The properties of the macrocyclic chelator-bearing derivatives make them potentially suitable for clinical purposes.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriAdependent primosome assembly is still a black box. Here, we report the 2.83 u AD naT 84–153 -dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.

c-Cbl-associated protein (CAP) is an important cytoskeletal adaptor protein involved in the regulation of adhesion turnover. The interaction between CAP and vinculin is critical for the recruitment of CAP to focal adhesions. The tandem SH3 domains (herein termed SH3a and SH3b) of CAP are responsible for its interaction with vinculin. However, the structural mechanism underlying the interaction between CAP and vinculin is poorly understood. In this manuscript, we report the solution structure of the tandem SH3 domains of CAP. Our NMR and ITC data indicate that the SH3a and SH3b domains of CAP simultaneously bind to a long proline-rich region of vinculin with different binding specificities. Furthermore, the crystal structures of the individual SH3a and SH3b domains complexed with their substrate peptides indicate that Q807(SH3a) and D881(SH3b) are the critical residues determining the different binding specificities of the SH3 domains. Based on the obtained structural information, a model of the SH3ab-vinculin complex was generated using MD simulation and SAXS data.

A publicar na Revista Chemistry a European Journal, Somatostatin-based radioligands were shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [55Co]DOTATOC (DOTATOC = 4,7, 10-tricarboxymethyl-1,4,7,10- tetraazacyclododecane-1-yl-acetyl-DPhe- Lys-Tyr-D-Trp-Lys-Thr-Lys-1- threoninol (disulfide bond) as a new radiopharmaceutical agent for PET was evaluated. 57Co was used as a surrogate of the positron emitter 55Co and the pharmacokinetics of [57Co]DOTATOC was investigated using two nude mouse models. The somatostatin receptor subtype (sst1-5) affinity profile of [natCo]DOTA-TOC was assessed using autoradiographic methods on membranes transfected with human somatostatin receptor subtypes. These studies revealed that [57Co]DOTATOC is an sst2-specific radiopeptide presenting the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing the AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst receptor-expressing tissues reflecting the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [57Co]DOTATOC and [67Ga]DOTATOC were discussed in terms of the structural parameters found for the chelate models CoII(DOTA)2- and GaIII(DOTA)-, whose X-ray structures were determined. Both chelates show sixfold coordination in pseudooctahedral arrangements.

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.