Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer.

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. METHODS: [77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. RESULTS: The maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13-0.24) nM and 0.46 (0.13-0.24) nM for PARP(+) and PARP(-) expressing cell lines, respectively. The EC50 was 0.27 (0.21-0.36) nM and 0.30 (0.22-0.41) nM for BRCA1(-) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3-9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. CONCLUSION: [77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

Intermetallic cobalt-gallium targets for production of germanium radioisotopes.

Increasing interest in targeted radionuclide therapy motivates the development of new radionuclides. The unique emission spectrum from 71Ge make it an ideal candidate for probing microdosimetric effects of low energy electrons absent confounding photon dose. This work reports a novel intermetallic target of Co and Ga for accelerator production of no-carrier-added 69/71Ge and a new method to isolate the Ge in high yields and purities.

Cyclotron production and radiochemical separation of 55Co and 58mCo from 54Fe, 58Ni and 57Fe targets.

This work presents the production with a cyclotron of the positron emitter 55Co via the 54Fe(d,n) and 58Ni(p,α) reactions and the Auger electron emitter 58mCo via the 57Fe(d,n) reaction after high current (40µA p and 60µA d) irradiation on electroplated targets. High specific activity radionuclides (up to 55.6 GBq/µmol 55Co and 31.8GBq/µmol 58mCo) with high radionuclidic purity (99.995% 55Co from 54Fe, 98.8% 55Co from 58Ni, and 98.7% 58mCo from 57Fe at end of bombardment, EoB), in high activity concentration (final separated radionuclide in < 0.6mL) and with almost quantitative overall activity separation yield (> 92%) were obtained after processing of the irradiated targets with novel radiochemical separation methods based on HCl dissolution and the resin N,N,N',N'-tetrakis-2-ethylhexyldiglycolamide (DGA, branched). One hour long irradiations using 38-65, 110-214 and 59-78mg of enriched 54Fe (99.93%), 58Ni (99.48%) and 57Fe (95.06%), respectively, electroplated over a 1.0cm2 surface, yielded 582 ± 66MBq 55Co, 372 ± 14MBq 55Co and 810 ± 186MBq 58mCo, respectively, decay corrected to EoB. The separation methods allow for the recovery of the costly enriched target materials, which were reconstituted into metallic targets after novel electroplating methods, with an overall recycling efficiency of 93 ± 4% for iron. The produced radionuclides were used to radiolabel the angiogenesis marker antibody TRC105 conjugated to the chelator NOTA as a demonstration of their quality.

Separation of 44Ti from proton irradiated scandium by using solid-phase extraction chromatography and design of 44Ti/44Sc generator system.

Scandium-44g (half-life 3.97h [1]) shows promise for positron emission tomography (PET) imaging of longer biological processes than that of the current gold standard, 18F, due to its favorable decay parameters. One source of 44gSc is the long-lived parent nuclide 44Ti (half-life 60.0 a). A 44Ti/44gSc generator would have the ability to provide radionuclidically pure 44gSc on a daily basis. The production of 44Ti via the 45Sc(p,2n) reaction requires high proton beam currents and long irradiation times. Recovery and purification of no-carrier added (nca) 44Ti from scandium metal targets involves complex separation chemistry. In this study, separation systems based on solid phase extraction chromatography were investigated, including branched diglycolamide (BDGA) resin and hydroxamate based ZR resin. Results indicate that ZR resin in HCl media represents an effective 44Ti/44gSc separation system.

Large scale accelerator production of 225Ac: Effective cross sections for 78-192MeV protons incident on 232Th targets.

Actinium-225 and 213Bi have been used successfully in targeted alpha therapy (TAT) in preclinical and clinical research. This paper is a continuation of research activities aiming to expand the availability of 225Ac. The high-energy proton spallation reaction on natural thorium metal targets has been utilized to produce millicurie quantities of 225Ac. The results of sixteen irradiation experiments of thorium metal at beam energies between 78 and 192MeV are summarized in this work. Irradiations have been conducted at Brookhaven National Laboratory (BNL) and Los Alamos National Laboratory (LANL), while target dissolution and processing was carried out at Oak Ridge National Laboratory (ORNL). Excitation functions for actinium and thorium isotopes, as well as for some of the fission products, are presented. The cross sections for production of 225Ac range from 3.6 to 16.7mb in the incident proton energy range of 78-192MeV. Based on these data, production of curie quantities of 225Ac is possible by irradiating a 5.0gcm-2 232Th target for 10 days in either BNL or LANL proton irradiation facilities.

Application of ion exchange and extraction chromatography to the separation of actinium from proton-irradiated thorium metal for analytical purposes.

Actinium-225 (t1/2=9.92d) is an α-emitting radionuclide with nuclear properties well-suited for use in targeted alpha therapy (TAT), a powerful treatment method for malignant tumors. Actinium-225 can also be utilized as a generator for (213)Bi (t1/2 45.6 min), which is another valuable candidate for TAT. Actinium-225 can be produced via proton irradiation of thorium metal; however, long-lived (227)Ac (t1/2=21.8a, 99% ß(-), 1% α) is co-produced during this process and will impact the quality of the final product. Thus, accurate assays are needed to determine the (225)Ac/(227)Ac ratio, which is dependent on beam energy, irradiation time and target design. Accurate actinium assays, in turn, require efficient separation of actinium isotopes from both the Th matrix and highly radioactive activation by-products, especially radiolanthanides formed from proton-induced fission. In this study, we introduce a novel, selective chromatographic technique for the recovery and purification of actinium isotopes from irradiated Th matrices. A two-step sequence of cation exchange and extraction chromatography was implemented. Radiolanthanides were quantitatively removed from Ac, and no non-Ac radionuclidic impurities were detected in the final Ac fraction. An (225)Ac spike added prior to separation was recovered at ≥ 98%, and Ac decontamination from Th was found to be ≥ 10(6). The purified actinium fraction allowed for highly accurate (227)Ac determination at analytical scales, i.e., at (227)Ac activities of 1-100 kBq (27 nCi to 2.7 µCi).

Regional and age-related differences in GAD67 expression of parvalbumin- and calbindin-expressing neurons in the rhesus macaque auditory midbrain and brainstem.

Neurons expressing the calcium binding proteins (CaBPs) parvalbumin (PV) and calbindin (CB) have shown age-related density changes throughout the ascending auditory system of both rodents and macaque monkeys. In the cerebral cortex, neurons expressing these CaBPs express markers of γ-aminobutyric acidergic neurotransmission, such as GAD67, and have well-understood physiological response properties. Recent evidence suggests that, in the rodent auditory brainstem, CaBP-containing cells do not express GAD67. It is unknown whether PV- and CB-containing cells in subcortical auditory structures of macaques similarly do not express GAD67, and a better understanding of the neurotransmission of neurons expressing these proteins is necessary for understanding the age-related changes in their density throughout the macaque auditory system. This was investigated with immunofluorescent double-labeling techniques to coregister PV- and CB-expressing neurons with GAD67 in the superior olivary complex and the inferior colliculus of young and aged rhesus macaques. The proportions of GAD67-expressing PV- and CB-positive neurons were computed with unbiased sampling techniques. Our results indicate that between 42% and 62% of PV- and CB-positive neurons in the auditory brainstem and midbrain express GAD67, which is significantly less than in the cerebrum. In general, fewer PV(+) neurons and more CB(+) neurons expressed GAD67 as a function of age. These results demonstrate that the inhibitory molecular profile of PV- and CB-expressing neurons can change with age in subcortical auditory structures and that these neurons are distinct from the well-described inhibitory interneurons that express these proteins in the cerebral cortex.

Increased T cell glucose uptake reflects acute rejection in lung grafts.

Although T cells are required for acute lung rejection, other graft-infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [(18)F]fluorodeoxyglucose ([(18)F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [(18)F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [(18)F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2-NBDG revealed that T cells, and in particular CD8(+) T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen-presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.

Cyclotron produced 44gSc from natural calcium.

(44g)Sc was produced by 16MeV proton irradiation of unenriched calcium metal with radionuclidic purity greater than 95%. The thick target yield at saturation for (44g)Sc was 213 MBq/µA, dwarfing the yields of contaminants (43)Sc,(44 m)Sc, (47)Sc and (48)Sc for practical bombardment times of 1-2h. Scandium was isolated from the dissolved calcium target by filtration, and reconstituted in small volumes of dilute HCl. Reactions with the chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) indicated a reactivity of 54 ± 14 Gbq/µmol at end-of-bombardment.

Very high specific activity 66/68Ga from zinc targets for PET.

This work describes the production of very high specific activity (66/68)Ga from (nat)Zn(p,n) and (66)Zn(p,n) using proton irradiations between 7 and 16 MeV, with emphasis on (66)Ga for use with common bifunctional chelates. Principal radiometallic impurities are (65)Zn from (p,x) and (67)Ga from (p,n). Separation of radiogallium from target material is accomplished with cation exchange chromatography in hydrochloric acid solution. Efficient recycling of Zn target material is possible using electrodeposition of Zn from its chloride form, but these measures are not necessary to achieve high specific activity or near-quantitative radiolabeling yields from natural targets. Inductively coupled plasma mass spectroscopy (ICP-MS) measures less than 2 ppb non-radioactive gallium in the final product, and the reactivity of (66)Ga with common bifunctional chelates, decay corrected to the end of irradiation, is 740 GBq/µmol (20 Ci/µmol) using natural zinc as a target material. Recycling enriched (66)Zn targets increased the reactivity of (66)Ga with common bifunctional chelates.

Cross sections of the 36Ar(d,α)34mCl, 40Ar(d,α)38Cl, and 40Ar(d,p)41Ar nuclear reactions below 8.4 MeV.

We have measured the cross section for production of the medically interesting isotope (34m)Cl, along with (38)Cl and (41)Ar, using deuteron bombardments of (36)Ar and (40)Ar below 8.4 MeV. ALICE/ASH analytical codes were employed to determine the shape of nuclear excitation functions, and experiments were performed using the University of Wisconsin tandem electrostatic accelerator to irradiate thin targets of argon gas.

WE-C-217BCD-04: Multimodality Imaging of Breast Cancer Experimental Lung Metastasis.

PURPOSE: Metastatic breast cancer (MBC) is incurable. The clinical gold standard for assessing tumor microvessel density (MVD), an independent prognostic marker in MBC, is CD 105 staining. The goal of this study is to develop a positron emission tomography (PET)/near-infrared fluorescent (NIRF) probe for imaging of CD105 expression in MBC (i.e. non-invasive measurement of MVD), as well as other applications such as early detection of metastasis, intraoperative guidance, etc. METHODS: TRC105, a chimeric anti-CD105 mAb, was dual-labeled with a NIRF dye and 89 Zr to yield 8 9 Zr-Df-TRC105-800CW. Luciferase-transfected 4T1 murine breast cancer cells were injected intravenously into female BALB/c mice to establish a lung MBC model. Bio luminescence imaging (BLI) was carried out to non- invasively monitor the lung tumor burden. Comprehensive in vivo/ex vivo studies were performed to investigate 8 9 Zr-Df-TRC105-800CW in this MBC model. Cetuximab was used as an isotype-matched control. RESULTS: Radiolabeled TRC105 has high tumor uptake in many tumor types in addition to MBC (e.g. pancreatic/prostate cancer and brain tumor), revealing broad clinical potential for TRC105-based agents. FACS analysis of HUVECs showed no difference in CD 105 binding between TRC105 and Df- TRC105-800CW. PET imaging revealed that 4T1 lung tumor uptake of 89 Zr-Df-TRC105-800CW was 8.7±1.4,10.9±0.5, and 9.7±1.1 %ID/g at 4, 24, and 48 h post-injection (n = 4), with excellent tumor contrast. Bio distribution studies, blocking, control studies with 8 9 Zr-Df-cetuximab- 800CW, ex vivo BLI/PET/NIRF imaging, and histology all confirmed CD 105 specificity of the tracer. NIRF imaging-guided removal of 4T1 tumors with Df-TRC105-800CW in a subcutaneous model was also straightforward. CONCLUSIONS: We report the first PET/NIRF imaging of CD105 expression in a MBC model. Broad clinical potential of TRC105- based agents was shown in many tumor types, which also enabled early detection of small metastases and provided intraoperative guidance for tumor removal.

WE-C-217BCD-06: In Vivo Targeting and Imaging of Tumor Vasculature with Radiolabeled, Antibody-Conjugated Nano-Graphene.

PURPOSE: Our goal was to explore nano-graphene for in vivo tumor targeting and quantitatively evaluate the pharmacokinetics and tumor targeting efficacy through PET imaging, using 6 4 Cu and 66 Ga as the radiolabel. METHODS: Nano-graphene oxide (GO) sheets, with amino group- terminated PEG chains (10 kDa) covalently attached, were conjugated to NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid, a suitable chelator for 6 4 Cu and 6 6 Ga) and TRC105 (a mAb that binds to CD 105, overexpressed on neovasculature). FACS analyses, size measurements, and serum stability studies were performed to characterize the GO conjugates before in vivo investigation (PET, bio distribution, blocking studies, etc.) in 4T1 murine breast tumor-bearing mice. Findings from imaging studies were then validated by histology. RESULTS: TRC105-conjugated GO, 20-30 nm in diameter, was specific for CD105 with little non-specific binding. Both 64 Cu- and 66 Ga- labled GO conjugates had excellent stability in mouse serum. Clearance of the GO conjugates in mice was via the hepatobiliary pathway. v Cu/6 6 Ga-NOTA-GO-TRC105 accumulated rapidly in the 4T1 tumor and tumor uptake remained stable over time (3.8±0.4, 4.5±0.4, 5.8±0.3, and 4.5±0.4 %ID/g at 0.5, 3, 7, and 24 h p.i. for66 Ga; 5.8±0.6, 5.3±0.6, 4.0±0.4, and3.4±0.1 %ID/g at 0.5, 3,24, and 48 h p.i. for 6 4 Cu; n = 4). Blocking studies confirmed CD105 specificity of 6 4 Cu/6 6 Ga-NOTA- GOTRC105, which was corroborated by bio distribution studies. Furthermore, microscopy examination of GO in light view mode and immunofluorescence staining revealed that targeting of NOTA-GO-TRC105 is tumor vasculature CD105 specific with little extravasation. CONCLUSIONS: For the first time, we demonstrated that GO can be specifically directed to the tumor neovasculature in vivo through targeting of CD105, a marker of tumor angiogenesis. The versatile chemistry of graphene-based nanomaterials makes them suitable nanoplatforms for future biomedical research, such as cancer theranostics.

The effect of rising vs. falling glucose level on amperometric glucose sensor lag and accuracy in Type 1 diabetes.

BACKGROUND: Because declining glucose levels should be detected quickly in persons with Type 1 diabetes, a lag between blood glucose and subcutaneous sensor glucose can be problematic. It is unclear whether the magnitude of sensor lag is lower during falling glucose than during rising glucose. METHODS: Initially, we analysed 95 data segments during which glucose changed and during which very frequent reference blood glucose monitoring was performed. However, to minimize confounding effects of noise and calibration error, we excluded data segments in which there was substantial sensor error. After these exclusions, and combination of data from duplicate sensors, there were 72 analysable data segments (36 for rising glucose, 36 for falling). We measured lag in two ways: (1) the time delay at the vertical mid-point of the glucose change (regression delay); and (2) determination of the optimal time shift required to minimize the difference between glucose sensor signals and blood glucose values drawn concurrently. RESULTS: Using the regression delay method, the mean sensor lag for rising vs. falling glucose segments was 8.9 min (95%CI 6.1-11.6) vs. 1.5 min (95%CI -2.6 to 5.5, P<0.005). Using the time shift optimization method, results were similar, with a lag that was higher for rising than for falling segments [8.3 (95%CI 5.8-10.7) vs. 1.5 min (95% CI -2.2 to 5.2), P<0.001]. Commensurate with the lag results, sensor accuracy was greater during falling than during rising glucose segments. CONCLUSIONS: In Type 1 diabetes, when noise and calibration error are minimized to reduce effects that confound delay measurement, subcutaneous glucose sensors demonstrate a shorter lag duration and greater accuracy when glucose is falling than when rising.

Specific α4ß2 nicotinic acetylcholine receptor binding of [F-18]nifene in the rhesus monkey.

OBJECTIVE: [F-18]Nifene is a PET radioligand developed to image α4ß2* nicotinic acetylcholine receptors (nAChR) in the brain. This work assesses the in vivo binding and imaging characteristics of [F-18]nifene in rhesus monkeys for the development of PET experiments examining nAChR binding. METHODS: Dynamic PET imaging experiments with [F-18]nifene were acquired in four anesthetized Macaca mulatta (rhesus) monkeys using a microPET P4 scanner. Data acquisition was initiated with a bolus injection of 109 ± 17 MBq [F-18]nifene and the time course of the radioligand in the brain was measured for up to 120 min. For two experiments, a displacement dose of (-)nicotine (0.03 mg kg(-1) , i.v.) was given 45-60 min post injection and followed 30 min later with a second [F-18]nifene injection to measure radioligand nondisplaceable uptake. Time activity curves were extracted in the regions of the antereoventral thalamus (AVT), lateral geniculate nucleus region (LGN), frontal cortex, and the cerebellum (CB). RESULTS: The highest levels of [F-18]nifene uptake were observed in the AVT and LGN. Target-to-CB ratios reached maximum values of 3.3 ± 0.4 in the AVT and 3.2 ± 0.3 in the LGN 30-45 min postinjection. Significant binding of [F-18]nifene was observed in the subiculum, insula cortex, temporal cortex, cingulate gyrus, frontal cortex, striatum, and midbrain areas. The (-)nicotine displaced bound [F-18]nifene to near background levels within 15 min postdrug injection. No discernable displacement was observed in the CB, suggesting its potential as a reference region. Logan graphical estimates using the CB as a reference region yielded binding potentials of 1.6 ± 0.2 in the AVT and 1.3 ± 0.1 in the LGN. The postnicotine injection displayed uniform nondisplaceable uptake of [F-18]nifene throughout gray and white brain matter. CONCLUSIONS: [F-18]Nifene exhibits rapid equilibration and a moderately high target to background binding profile in the α4ß2* nAChR rich regions of the brain, thus providing favorable imaging characteristics as a PET radiotracer for nAChR assay.

In vivo kinetics of [F-18]MEFWAY: a comparison with [C-11]WAY100635 and [F-18]MPPF in the nonhuman primate.

UNLABELLED: [F-18]Mefway was developed to provide an F-18 labeled positron emission tomography (PET) neuroligand with high affinity for the serotonin 5-HT(1A) receptor to improve the in vivo assessment of the 5-HT(1A) system. The goal of this work was to compare the in vivo kinetics of [F-18]mefway, [F-18]MPPF, and [C-11]WAY100635 in the rhesus monkey. METHODS: Each of four monkeys were given bolus injections of [F-18]mefway, [C-11]WAY100635, and [F-18]MPPF and scans were acquired with a microPET P4 scanner. Arterial blood was sampled to assay parent compound throughout the time course of the PET experiment. Time activity curves were extracted in the high 5-HT(1A) binding areas of the anterior cingulate cortex (ACG), mesial temporal cortex, raphe nuclei, and insula cortex. Time activity curves were also extracted in the cerebellum, which was used as a reference region. The in vivo kinetics of the radiotracers were compared based on the nondisplaceable distribution volume (V(ND) ) and binding potential (BP(ND) ). RESULTS: At 30 min, the fraction of radioactivity in the plasma due to parent compound was 19%, 28%, and 29% and cleared from the arterial plasma at rates of 0.0031, 0.0078, and 0.0069 (min⁻¹) ([F-18]mefway, [F-18]MPPF, [C-11]WAY100635). The BP(ND) in the brain regions were mesial temporal cortex: 7.4 ± 0.6, 3.1 ± 0.4, 7.0 ± 1.2, ACG: 7.2 ± 1.2, 2.1 ± 0.2, 7.9 ± 1.2; raphe nuclei: 3.7 ± 0.6, 1.3 ± 0.3, 3.3 ± 0.7; and insula cortex: 4.2 ± 0.6, 1.2 ± 0.1, 4.7 ± 1.0 for [F-18]mefway, [F-18]MPPF, and [C-11]WAY100635 respectively. CONCLUSIONS: In the rhesus monkey, [F-18]mefway has similar in vivo kinetics to [C-11]WAY100635 and yields greater than 2-fold higher BP(ND) than [F-18]MPPF. These properties make [F-18]mefway a promising radiotracer for 5-HT(1A) assay, providing higher counting statistics and a greater dynamic range in BP(ND).

Eelgrass meadows in the California Channel Islands and adjacent coast reveal a mosaic of two species, evidence for introgression and variable clonality.

BACKGROUND AND AIMS: Seagrasses are important facilitator species in shallow, soft-bottom marine environments worldwide and, in many places, are threatened by coastal development and eutrophication. One narrow-leaved species (Zostera marina) and one wide-leaved species, variously designated as Z. marina, Z. pacifica or Z. asiatica, are found off the California Channel Islands and adjacent California-Mexico coast. The aim of the present study was to confirm species identification genetically and to link patterns of genetic diversity, connectivity and hybridization among and within the populations with historical sea levels (Ice Age) or the contemporary environment. METHODS: Samples (n = 11-100) were collected from 28 sites off five California Channel Islands and six sites off the adjacent coast of southern California and Baja California, Mexico. DNA polymorphisms of the rDNA-ITS (internal transcribed spacer) cistron (nuclear), the matK intron (chloroplast) and nine microsatellite loci (nuclear) were examined in a population genetic and phylogeographic context. KEY RESULTS: All wide-leaved individuals were Z. pacifica, whereas narrow-leaved forms were Z. marina. Microsatellite genotypes were consistent with hybridization between the two species in three populations. The present distribution of Z. pacifica follows a glacial age land mass rather than present oceanographic regimes, but no link was observed between the present distribution of Z. marina and past or present environments. Island populations of Z. marina often were clonal and characterized by low genotypic diversity compared with populations along the Baja California coast. The high level of clonal connectivity around Santa Catalina Island indicated the importance of dispersal and subsequent re-establishment of vegetative fragments. CONCLUSIONS: The pristine environmental conditions of offshore islands do not guarantee maximum genetic diversity. Future restoration and transplantation efforts of seagrasses must recognize cryptic species and consider the degree of both genetic and genotypic variation in candidate donor populations.

Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1.

We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung. However, ADAMTS9 is highly expressed in embryonic and adult tissues, and therefore we characterized the ADAMTS-9 protein further. Although the ADAMTS-9 zymogen has many proprotein convertase processing sites, pulse-chase analysis, site-directed mutagenesis, and amino acid sequencing demonstrated that maturation to the active form occurs by selective proprotein convertase (e.g. furin) cleavage of the Arg(287)-Phe(288) bond. Although lacking a transmembrane sequence, ADAMTS-9 is retained near the cell surface as well as in the ECM of transiently transfected COS-1 and 293 cells. COS-1 cells transfected with ADAMTS9 (but not vector-transfected cells) proteolytically cleaved bovine versican and aggrecan core proteins at the Glu(441)-Ala(442) bond of versican V1 and the Glu(1771)-Ala(1772) bond of aggrecan, respectively. In contrast, the ADAMTS-9 catalytic domain alone was neither localized to the cell surface nor able to confer these proteolytic activities on cells, demonstrating that the ancillary domains of ADAMTS-9, including the TSRs, are required both for specific extracellular localization and for its versicanase and aggrecanase activities.

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.