Parallel studies of the preparation of Re and (99m)Tc agents aid in interpreting the nature of tracer (99m)Tc radiopharmaceuticals. Aqueous solutions of the fac-[(99m)Tc(CO)(3)(H(2)O)(3)](+) cation are gaining wide use and are readily prepared, but such solutions of the fac-[Re(CO)(3)(H(2)O)(3)](+) cation (1) are not so easily accessible. Herein we describe a new, reliable, and straightforward preparation of aqueous solutions of 1, characterized by HPLC and ESI-MS. Treatment of solutions of 1 with thioether-bearing amino acids, AAH = S-methyl-l-cysteine (MECYSH), S-propyl-l-cysteine (PRCYSH), and methionine (METH), gave high yields of fac-Re(CO)(3)AA complexes. X-ray crystallographic and NMR analyses indicated that MECYS(-), PRCYS(-), and MET(-) were bound in fac-Re(CO)(3)AA complexes as tridentate monoanionic ligands through amino, thioether, and alpha-carboxyl groups. In CD(3)OD, (1)H NMR spectra have broad signals but have two sets of signals at -10 degrees C, consistent with two isomers with different configurations at the pyramidal sulfur; these interconvert slowly on the NMR time scale at low temperatures. Indeed, the crystal structure of the fac-Re(CO)(3)(PRCYS) reveals a mixture of the two possible diastereoisomers. S-(Carboxymethyl)-l-cysteine (CCMH(2)) and 1 gave two products, 5A (kinetically favored) and 5B (thermodynamically favored). X-ray crystallographic analyses of a crystal of 5B and of a 1:1 cocrystal of 5A and 5B showed that 5A and 5B are diastereoisomers with the CCMH(-) alpha-carboxyl group dangling. In addition to the amino and thioether groups, the S-(carboxymethyl) carboxyl group is coordinated, a feature that slows interconversion of diastereoisomers relative to the other fac-Re(CO)(3)AA complexes because interconversion can now occur only after the rupture of Re-ligand bonds. These N, O, and S tridentate adducts are quite stable, and the grouping has promise in (99m)Tc(CO)(3) tracer development.

To identify new genetic risk factors for cervical cancer, we conducted a genome-wide association study in the Han Chinese population. The initial discovery set included 1,364 individuals with cervical cancer (cases) and 3,028 female controls, and we selected a 'stringently matched samples' subset (829 cases and 990 controls) from the discovery set on the basis of principal component analysis; the follow-up stages included two independent sample sets (1,824 cases and 3,808 controls for follow-up 1 and 2,343 cases and 3,388 controls for follow-up 2). We identified strong evidence of associations between cervical cancer and two new loci: 4q12 (rs13117307, Pcombined, stringently matched=9.69×10(-9), per-allele odds ratio (OR)stringently matched=1.26) and 17q12 (rs8067378, Pcombined, stringently matched=2.00×10(-8), per-allele ORstringently matched=1.18). We additionally replicated an association between HLA-DPB1 and HLA-DPB2 (HLA-DPB1/2) at 6p21.32 and cervical cancer (rs4282438, Pcombined, stringently matched=4.52×10(-27), per-allele ORstringently matched=0.75). Our findings provide new insights into the genetic etiology of cervical cancer.

The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226 -299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding. Varicella zoster virus (VZV)1 is a member of the alphaherpesvirinae and the causative agent of chicken pox (varicella) and shingles (zoster). The VZV genome is a linear doublestranded DNA molecule, which encodes approximately seventy proteins (1). The entire complement of VZV genes is believed to be expressed during lytic infection in three broad kinetic classes, immediate early (IE), early (E), and late (L). Transcription of VZV genes is performed by the host cell RNA polymerase II, as is the case with all other herpes viruses. Efficient expression of the VZV genome is driven by a small group of VZV gene products including those encoded by open reading frames (ORFs) 62, 4, 61, 63, and 10 (2-11). The major viral transactivator is the product of ORF 62 and its complement, ORF 71, which lie within the inverted repeats bracketing the Us region of VZV DNA. This protein is commonly designated IE62 since it is synthesized in the immediate early phase of lytic VZV gene expression. IE62 contains a potent N-terminal acidic transactivation domain and is capable of activating the expression of all three kinetic classes of VZV genes (12)(13)(14).While IE62 is involved in transactivation of VZV promoters, careful analysis of a limited number of individual viral promoters has shown that cellular transcription factors acting at sites upstream of the coding regions of the viral genes are also involved in the mechanism of IE62 activation. These proteins include the ubiquitous, sequence specific cellular factor Sp1. Sp1 is the protoype of a family of closely related factors which bind to GC-rich elements including the GC-box (GGGCGG or GGGCGGG) and the related GT/CACCC-box. Sp1 contains five distinct domains, four of which (A, B, C, and D) are involved in various aspects of transcriptional activation as well as a DNA binding region conta...

Varicella-zoster virus (VZV) glycoprotein I is dispensable in cell culture but necessary for infection of human skin and T cells in

The coordination chemistry of lanthionine (LANH2) and cystathionine (CSTH2) dipeptides, which respectively consist of two cysteines and one cysteine and one homocysteine linked by a thioether bridge, is almost unstudied. Recently for fac-[99mTc(CO)3(LAN)]- isomers, the first small 99mTc(CO)3 agents evaluated in humans were found to give excellent renal images and to have a high specificity for renal excretion. Herein we report the synthesis and characterization of Re complexes useful for interpreting the nature of tracer 99mTc radiopharmaceuticals. Treatment of [Re(CO)3(H2O)3]OTf with commercially available LANH2 (a mixture of meso (d,l) and chiral (dd,ll) isomers) gave three HPLC peaks, 1A, 1B, and 1C, but treatment with CSTH2 (l,l isomer) gave one major product, Re(CO)3(CSTH) (2). Crystalline Re(CO)3(LANH) products were best obtained with synthetic LANH2, richer in meso or chiral isomers. X-ray crystallography showed that these dipeptides coordinate as tridentate N2S-bound ligands with two dangling carboxyls. The LANH ligand is meso in 1A and 1C and chiral in 1B. While 1A (kinetically favored) is stable at ambient temperature for days, it converted into 1C (thermodynamically favored) at 100 degrees C; after 6 h, equilibrium was reached at a 1A:1C ratio of 1:2 at pH 8. The structures provide a rationale for this behavior and for the fact that 1A and 1C have simple NMR spectra. This simplicity results from fluxional interchange between an enantiomer with both chelate rings having the same delta pucker and an enantiomer with both chelate rings having the same lambda pucker. Agents with the [99mTc(CO)3]+ core and N2S ligands show promise of becoming an important class of 99mTc radiopharmaceuticals. The chemistry of Re analogues with these ligands, such as the LAN2- complexes reported here, provides a useful background for designing new small agents and also tagged large agents because two uncoordinated carboxyl groups are available for conjugation with biological molecules such as proteins.

Background and Objective: Endometrial cancer (EC) is a common gynecological malignancy worldwide. Despite advances in the development of strategies for treating EC, prognosis of the disease remains unsatisfactory, especially for advanced EC. The aim of this study was to identify novel genes that can be used as potential biomarkers for identifying the prognosis of EC and to construct a novel risk stratification using these genes. Methods and Results: An mRNA sequencing dataset, corresponding survival data and expression profiling of an array of EC patients were obtained from The Cancer Genome Atlas and Gene Expression Omnibus, respectively. Common differentially expressed genes (DEGs) were identified based on sequencing and expression as given in the profiling dataset. Pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization, and Integrated Discovery. The protein-protein interaction network was established using the string online database in order to identify hub genes. Univariate and multivariable Cox regression analyses were used to screen prognostic DEGs and to construct a prognostic signature. Survival analysis based on the prognostic signature was performed on TCGA EC dataset. A total of 255 common DEGs were found and 11 hub genes (TOP2A, CDK1, CCNB1, CCNB2, AURKA, PCNA, CCNA2, BIRC5, NDC80, CDC20, and BUB1BA) that may be closely related to the pathogenesis of EC were identified. A panel of 7 DEG signatures consisting of PHLDA2, GGH, ESPL1, FAM184A, KIAA1644, ESPL1, and TRPM4 were constructed. The signature performed well for prognosis prediction (p < 0.001) and time-dependent receiver-operating characteristic (ROC) analysis displayed an area under the curve (AUC) of 0.797, 0.734, 0.729, and 0.647 for 1, 3, 5, and 10-year overall survival (OS) prediction, respectively. Conclusion: This study identified potential genes that may be involved in the pathophysiology of EC and constructed a novel gene expression signature for EC risk stratification and prognosis prediction.

We study Re analogues of 99mTc renal agents to interpret previous results at the 99mTc tracer level. The relative propensities of amine donors vs. carboxylate oxygen donors of four L = polyaminocarboxylate ligands to coordinate in fac-[ReI(CO)3L]n complexes were assessed by examining the reaction of fac-[ReI(CO)3(H2O)3]+ under conditions differing in acidity and temperature. All four L [N,N-bis-(2-aminoethyl)glycine (DTGH), N,N-ethylenediaminediacetic acid, diethylenetriamine-N-malonic acid, and diethylenetriamine-N-acetic acid] can coordinate as tridentate ligands while creating a dangling chain terminated in a carboxyl group. Dangling carboxyl groups facilitate renal clearance in fac-[99mTcI(CO)3L]n agents. Under neutral conditions, the four ligands each gave two fac-[ReI(CO)3L]n products with HPLC traces correlating well with known traces of the fac-[99mTcI(CO)3L]n mixtures. Such mixtures are common in renal agents because the needed dangling carboxyl group can compete for a coordination site. However, the HPLC separations needed to assess the biodistribution of a single tracer are impractical in a clinical setting. One goal in investigating this Re chemistry is to identify conditions for avoiding this problem of mixtures in preparations of fac-[99mTcI(CO)3L]n renal tracers. After separation and isolation of the fac-[ReI(CO)3L]n products, NMR analysis of all products and single crystal X-ray crystallographic analysis of both DTGH products as well as one product each from the other L allowed us to establish coordination mode unambiguously. The product favored in acidic conditions has a dangling amine chain and more bound oxygen. The product favored in basic conditions has a dangling carboxyl chain and more bound nitrogen. At the elevated temperatures used for simulating tracer preparation, equilibration was facile (ca. one hour or less), allowing selective formation of one product by utilizing acidic or basic conditions. The results of this fundamental study offer protocols and guidance useful for the design and preparation of fac-[99mTcI(CO)3L]n agents consisting of a single tracer.

Introduction:The first human studies of a characterized radiopharmaceutical containing a { 99m Tc (CO) 3 } + core, Na[ 99m Tc(CO) 3 (LAN)], demonstrated that Na[ 99m Tc(CO) 3 (LAN)] was an excellent renal imaging agent however, its clearance was less than that of 131 Iorthoiodohippurate ( 131 I-OIH) and it did not provide a direct measure of effective renal plasma flow. In order to develop a 99m Tc renal agent with pharmacokinetic properties equivalent to those of 131 I-OIH, we investigated the 99m Tc(CO) 3 /Re(CO) 3 complexes formed from carboxymethylmercaptosuccinic acid (CMSAH 3 ) and thiodisuccinic acid (TDSAH 4 ). Once the ligand is bound to 99m Tc(CO) 3 through a thioether and two carboxyl groups, the complexes have at least one unbound carboxyl group, essential for the interaction with the renal tubular transporter.Methods: X-ray crystal structural analysis of NMe 4 [Re(CO) 3 (CMSAH)] was performed to interpret the nature of 99m Tc tracers. CMSAH 3 and TDSAH 4 were radiolabeled by incubating each ligand and the precursor [ 99m Tc(CO) 3 (H 2 O) 3 ] + at 70 °C (pH 7) for 30 min. The products were purified by RP-HPLC and biodistribution studies were performed in Sprague-Dawley rats, with 131 I-OIH as an internal control at 10 and 60 min.Results: Radiolabeling CMSAH 3 and TDSAH 4 with the [ 99m TcCO) 3 (H 2 O) 3 ] + precursor gave products quantitatively. Analysis of the Re(CO) 3 complexes with the CMSAH 3 and TDSAH 4 ligands demonstrates that ligands are bound in 99m Tc /Re(CO) 3 complexes through a thioether and two deprotonated carboxyl groups (forming tridentate dianionic moieties, generally with two fivemembered chelate rings). Renal excretion at 60 min (activity in the urine as a percent of 131 I-OIH) was 68 ± 1% for Na 3 [ 99m Tc(CO) 3 (TDSA)], but was 98 ± 1% for Na 2 [ 99m Tc(CO) 3 (CMSA)]. Conclusion:In rats, Na 2 [ 99m Tc(CO) 3 (CMSA)] is extracted by the kidneys and eliminated in the urine almost as rapidly as 131 I-OIH; consequently Na 2 [ 99m Tc(CO) 3 (CMSA)] may provide a direct measure of effective renal plasma flow and further evaluation in humans is warranted.